Biologically active peptides from funcional domains of bactericidal/permeability-increasing protein and uses thereof

ABSTRACT

The present invention provides peptides having an amino acid sequence that is the amino acid sequence of a human bactericidal/permeability-increasing protein (BPI) functional domain or a subsequence thereof, and variants of the sequence or subsequence thereof, having at least one of the BPI biological activities, such as heparin binding, heparin neutralization, LPS binding, LPS neutralization or bactericidal activity. The invention provides peptides and pharmaceutical compositions of such peptides for a variety of therapeutic uses.

This is a continuation of application U.S. Ser. No. 08/209,762, filedMar. 11, 1994, which is a continuation-in-part of application Ser. No.08/183,222, filed Jan. 14, 1994 (now abandoned), which is acontinuation-in-part of application Ser. No. 08/093,202, filed Jul. 15,1993 (now abandoned), which is a continuation-in-part of applicationSer. No. 08/030,644, filed Mar. 12, 1993 (issued as U.S. Pat. No.5,348,942).

BACKGROUND OF THE INVENTION

The present invention relates to peptides derived from or based onbactericidal/permeability-increasing protein and therapeutic uses ofsuch peptides.

Bactericidal/permeability-increasing protein (BPI) is a protein isolatedfrom the granules of mammalian polymorphonuclear neutrophils (PMNs),which are blood cells essential in defending a mammal against invadingmicroorganisms. Human BPI has been isolated from PMNs by acid extractioncombined with either ion exchange chromatography (Elsbach, 1979, J.Biol. Chem. 254: 11000) or E. coli affinity chromatography (Weiss etal., 1987, Blood 69: 652), and has potent bactericidal activity againsta broad spectrum of Gram-negative bacteria. The molecular weight ofhuman BPI is approximately 55,000 daltons (55 kD). The complete aminoacid sequence of human BPI, as well as the nucleotide sequence of DNAencoding BPI, have been elucidated by Gray et al., 1989, J. Biol. Chem.264: 9505, incorporated herein by reference (see FIG. 1 in Gray et al.).

The bactericidal effect of BPI has been shown to be highly specific tosensitive Gram-negative species. The precise mechanism by which BPIkills Gram-negative bacteria is not yet known, but it is known that BPImust first attach to the surface of susceptible Gram-negative bacteria.This initial binding of BPI to the bacteria involves electrostaticinteractions between BPI, which is a basic, (i.e., positively charged)protein, and negatively charged sites on lipopolysaccharides (LPS). LPSis also known as "endotoxin" because of the potent inflammatory responsethat it stimulates. LPS induces the release of mediators by hostinflammatory cells which may ultimately result in irreversible endotoxicshock. BPI binds to Lipid A, the most toxic and most biologically activecomponent of LPS.

BPI is also capable of neutralizing the endotoxic properties of LPS towhich it binds. Because of its Gram-negative bactericidal properties andits ability to bind to and neutralize LPS, BPI can be utilized for thetreatment of mammals suffering from diseases caused by Gram-negativebacteria, including bacteremia, endotoxemia, and sepsis. These dualproperties of BPI make BPI particularly useful and advantageous for suchtherapeutic administration.

A proteolytic fragment corresponding to the amino-terminal portion ofhuman BPI possesses the LPS binding and neutralizing activities andantibacterial activity of the naturally-derived 55 kD human holoprotein.In contrast to the amino-terminal portion, the carboxyl-terminal regionof isolated human BPI displays only slightly detectable antibacterialactivity (Ooi et al., 1991, J. Exp. Med. 174: 649). One BPIamino-terminal fragment, comprising approximately the first 199 aminoacid residues of the human BPI holoprotein and referred to as "rBPI₂₃ "(see Gazzano-Santoro et al., 1992, Infect. Immun. 60: 4754-4761) hasbeen produced by recombinant means as a 23 kD protein. rBPI₂₃ has beenintroduced into human clinical trials. Proinflammatory responses toendotoxin were significantly ameliorated when rBPI₂₃ was co-administeredwith LPS.

Other endotoxin binding and neutralizing peptides are known in the art.One example is Limulus antilipopolysaccharide factor (LALF) fromhorseshoe crab amebocytes (Warren et al., 1992, Infect. Immunol. 60:2506-2513). Another example is a cyclic, cationic lipopeptide fromBacillus polymyxa, termed Polymyxin B₁. Polymyxin B₁ is composed of sixα,γ-diaminobutyric acid residues, one D-phenylalaline, one leucine, onethreonine and a 6-methyloctanoyl moiety (Morrison and Jacobs, 1976,Immunochem. 13: 813-818) and is also bactericidal. Polymyxin analogueslacking the fatty acid moiety are also known, which analogues retain LPSbinding capacity but are without appreciable bactericidal activity(Danner et al., 1989, Antimicrob. Agents Chemother. 33: 1428-1434).Similar properties have also been found with synthetic cyclizedpolymyxin analogues (Rustici et al., 1993, Science 259: 361-365).

Known antibacterial peptides include cecropins and magainins. Thececropins are a family of antibacterial peptides found in the hemolymphof lepidopteran insects (Wade et al., 1990, Proc. Natl. Acad. Sci.U.S.A. 87: 4761-4765), and the magainins are a family of antibacterialpeptides found in Xenopus skin and gastric mucosa (Zasloff et al., 1988,Proc. Natl. Acad. Sci. U.S.A. 85: 910-913). These peptides are linearand range from about 20 to about 40 amino acids in length. A less activemammalian cecropin has been reported from porcine intestinal mucosa,cecropin P1 (Boman et al., 1993, Infect. Immun. 61: 2978-2984). Thececropins are generally reported to be more potent than the magainins inbactericidal activity but appear to have less mammalian cellcytotoxicity. The cecropins and magainins are characterized by acontinuous, amphipathic α-helical region which is necessary forbactericidal activity. The most potent of the cecropins identified todate is cecropin A. The sequence of the first ten amino acids of thececropin A has some homology with the BPI amino acid sequence 90-99.However, the other 27 amino acids of cecropin A are clearly necessaryfor its bactericidal activity and there is little homology with BPI forthose 27 amino acids. The magainins have even less homology with the BPIsequence.

Of interest to the present application are the disclosures in PCTInternational Application PCT/US91/05758 relating to compositionscomprising BPI and an anionic compound, which compositions are said toexhibit (1) no bactericidal activity and (2) endotoxin neutralizingactivity. Anionic compounds are preferably a protein such as serumalbumin but can also be a polysaccharide such as heparin. In addition,Weiss et al. (1975, J. Clin. Invest. 55: 33-42) disclose that heparinsulfate and LPS block expression of the permeability-increasing activityof BPI. However, neither reference discloses that BPI actuallyneutralizes the biologic activities of heparin. Heparin binding does notnecessarily imply heparin neutralization. For example, a family ofheparin binding growth factors (HBGF) requires heparin as a cofactor toelicit a biological response. Examples of HBGF's include: fibroblastgrowth factors (FGF-1, FGF-2) and endothelial cell growth factors(ECGF-1, ECGF-2). Antithrombin III inhibition of clotting cascadeproteases is another example of a heparin binding protein that requiresheparin for activity and clearly does not neutralize heparin. Heparinbinding proteins that do neutralize heparin (e.g., platelet factor IV,protamine, and thrombospondin) are generally inhibitory of theactivities induced by heparin binding proteins that use heparin as acofactor.

BPI (including amino-terminal fragments thereof) has a number of otherimportant biological activities. For example, BPI has been shown to haveheparin binding and heparin neutralization activities in copending andco-assigned parent U.S. patent application Ser. No. 08/030,644 filedMar. 12, 1993 and continuation-in-part U.S. patent application Ser. No.08/093,202, filed Jul. 15, 1993, the disclosures of which areincorporated by reference herein. These heparin binding andneutralization activities of BPI are significant due to the importanceof current clinical uses of heparin. Heparin is commonly administered indoses of up to 400 U/kg during surgical procedures such ascardiopulmonary bypass, cardiac catherization and hemodialysisprocedures in order to prevent blood coagulation during such procedures.When heparin is administered for anticoagulant effects during surgery,it is an important aspect of post-surgical therapy that the effects ofheparin are promptly neutralized so that normal coagulation function canbe restored. Currently, protamine is used to neutralize heparin.Protamines are a class of simple, arginine-rich, strongly basic, lowmolecular weight proteins. Administered alone, protamines (usually inthe form of protamine sulfate) have anti-coagulant effects. Whenadministered in the presence of heparin, a stable complex is formed andthe anticoagulant activity of both drugs is lost. However, significanthypotensive and anaphylactoid effects of protamine have limited itsclinical utility. Thus, due to its heparin binding and neutralizationactivities, BPI has potential utility as a substitute for protamine inheparin neutralization in a clinical context without the deleteriousside-effects which have limited the usefulness of the protamines. Theadditional antibacterial and anti-endotoxin effects of BPI would also beuseful and advantageous in post-surgical heparin neutralization comparedwith protamine.

Additionally, BPI is useful in inhibiting angiogenesis due in part toits heparin binding and neutralization activities. In adults, angiogenicgrowth factors are released as a result of vascular trauma (woundhealing), immune stimuli (autoimmune disease), inflammatory mediators(Prostaglandins) or from tumor cells. These factors induce proliferationof endothelial cells (which is necessary for angiogenesis) via aheparin-dependent receptor binding mechanism (see Yayon et al., 1991,Cell 64: 841-848). Angiogenesis is also associated with a number ofother pathological conditions, including the growth, proliferation, andmetastasis of various tumors; diabetic retinopathy, retrolentalfibroplasia, neovascular glaucoma, psoriasis, angiofibromas, immune andnon-immune inflammation including rheumatoid arthritis, capillaryproliferation within atherosclerotic plaques, hemangiomas, endometriosisand Kaposi's sarcoma. Thus, it would be desirable to inhibitangiogenesis in these and other instances, and the heparin binding andneutralization activities of BPI are useful to that end.

Several other heparin neutralizing proteins are also known to inhibitangiogenesis. For example, protamine is known to inhibittumor-associated angiogenesis and subsequent tumor growth see Folkman etal., 1992, Inflammation: Basic Principles and Clinical Correlates, 2ded., (Galin et al., eds., Review Press, N.Y.), Ch. 40, pp. 821-839! Asecond heparin neutralizing protein, platelet factor IV, also inhibitsangiogenesis (i.e., is angiostatic). Collagenase inhibitors are alsoknown to inhibit angiogenesis (see Folkman et al., 1992, ibid.) Anotherknown angiogenesis inhibitor, thrombospondin, binds to heparin with arepeating serine/tryptophan motif instead of a basic amino acid motif(see Guo et al., 1992, J. Biol. Chem. 267: 19349-19355).

Another utility of BPI involves pathological conditions associated withchronic inflammation, which is usually accompanied by angiogenesis. Oneexample of a human disease related to chronic inflammation is arthritis,which involves inflammation of peripheral joints. In rheumatoidarthritis, the inflammation is immune-driven, while in reactivearthritis, inflammation is associated with infection of the synovialtissue with pyogenic bacteria or other infectious agents. Folkman etal., 1992, supra, have also noted that many types of arthritis progressfrom a stage dominated by an inflammatory infiltrate in the joint to alater stage in which a neovascular pannus invades the joint and beginsto destroy cartilage. While it is unclear whether angiogenesis inarthritis is a causative component of the disease or an epiphenomenon,there is evidence that angiogenesis is necessary for the maintenance ofsynovitis in rheumatoid arthritis. One known angiogenesis inhibitor,AGM1470, has been shown to prevent the onset of arthritis and to inhibitestablished arthritis in collagen-induced arthritis models (Peacock etal., 1992, J. Exp. Med. 175: 1135-1138). While nonsteroidalanti-inflammatory drugs, corticosteroids and other therapies haveprovided treatment improvements for relief of arthritis, there remains aneed in the art for more effective therapies for arthritis and otherinflammatory diseases.

There continues to exist a need in the art for new products and methodsfor use as bactericidal agents and endotoxin neutralizing agents, andfor heparin neutralization and inhibition of angiogenesis (normal orpathological). One avenue of investigation towards fulfiling this needis the determination of the functional domains of the BPI proteinspecifying each of these biological activities. Advantageous therapeuticembodiments would therefore comprise BPI functional domain peptideshaving one or more than one of the activities of BPI.

SUMMARY OF THE INVENTION

This invention provides small, readily-produced peptides having an aminoacid sequence that is the amino acid sequence of a BPI functional domainor a subsequence thereof and variants of the sequence or subsequencehaving at least one of the biological activities of BPI, such as heparinbinding, heparin neutralization, LPS binding, LPS neutralization orbactericidal activity. The functional domains of BPI discovered anddescribed herein include: domain I, encompassing the amino acid sequenceof BPI from about amino acid 17 to about amino acid 45; domain II,encompassing the amino acid sequence of BPI from about amino acid 65 toabout amino acid 99; and domain III, encompassing the amino acidsequence of BPI from about amino acid 142 to about amino acid 169. Thus,the BPI functional domain peptides are based on the amino-terminalportion of human BPI.

The peptides of the invention include linear and cyclized peptides, andpeptides that are linear, cyclized and branched-chain combinations ofparticular BPI functional domain amino acid sequences or subsequencesthereof and variants of the sequence or subsequence. Combinationpeptides include peptides having the sequence or subsequence andvariants of the sequence or subsequence of the same or differentfunctional domains of BPI that are covalently linked together.Specifically included are combinations from two to about 10 peptides ofany particular sequence or subsequence thereof and variants of thatsequence or subsequence. The invention also provides peptides havingadditional biological activities distinct from the known biologicalactivities of BPI, including but not limited to bactericidal activityhaving an altered target cell species specificity. Peptides havingparticular biological properties of BPI that are enhanced or decreasedcompared with the biological properties of BPI are also provided.

The peptides of the invention include linear and cyclized peptides, andpeptides that are linear, cyclized and branched-chain amino acidsubstitution and additional variants of particular BPI functional domainamino acid sequences or subsequences thereof. For the substitutionvariants, amino acid residues at one or more positions in each of thepeptides are replaced with a different amino acid residue (includingatypical amino acid residues) from that found in the correspondingposition of the BPI functional domain from which the specific peptide isderived. For the addition variants, peptides may include up to about atotal of 10 additional amino acids, covalently linked to either theamino-terminal or carboxyl-terminal extent, or both, of the BPIfunctional domain peptides herein described. Such additional amino acidsmay duplicate amino acids in BPI contiguous to a functional domain ormay be unrelated to BPI amino acid sequences and may include atypicalamino acids. Linear, cyclized, and branched-chain combinationembodiments of the amino acid substitution and addition variant peptidesare also provided as peptides of the invention, as are cyclizedembodiments of each of the aforementioned BPI functional domainpeptides. In addition, peptides of the invention may be provided asfusion proteins with other functional targeting agents, such asimmunoglobulin fragments. Addition variants include derivatives andmodifications of amino acid side chain chemical groups such as amines,carboxylic acids, alkyl and phenyl groups.

The invention provides pharmaceutical compositions for use in treatingmammals for neutralizing endotoxin, killing Gram-negative andGram-positive bacteria and fungi, neutralizing the anticoagulantproperties of heparin, inhibiting angiogenesis, inhibiting tumor andendothelial cell proliferation, and treating chronic inflammatorydisease states. The pharmaceutical compositions comprise unit dosages ofthe BPI peptides of this invention in solid, semi-solid and liquiddosage forms such as tablet pills, powder, liquid solution orsuspensions and injectable and infusible solutions.

This invention provides peptides having an amino acid sequence which isthe amino acid sequence of human BPI from about position 17 to aboutposition 45 comprising functional domain I, having the sequence:##STR1## and subsequences thereof which have biological activity,including but not limited to one or more of the activities of BPI, forexample, bactericidal activity, LPS binding, LPS neutralization, heparinbinding or heparin neutralization. Also provided in this aspect of theinvention are peptides having substantially the same amino acid sequenceof the functional domain I peptides having the amino acid sequence ofBPI from about position 17 to about position 45 or subsequences thereof.Additionally, the invention provides peptides which contain two or moreof the same or different domain I peptides or subsequence peptidescovalently linked together.

This invention provides peptides having an amino acid sequence which isthe amino acid sequence of human BPI from about position 65 to aboutposition 99 comprising functional domain II, having the sequence:##STR2## and subsequences thereof which have biological activity,including but not limited to one or more of the activities of BPI, forexample, bactericidal activity, LPS binding, LPS neutralization, heparinbinding or heparin neutralization. Also provided in this aspect of theinvention are peptides having substantially the same amino acid sequenceof the functional domain II peptides having the amino acid sequence ofBPI from about position 65 to about position 99 or subsequences thereof.Additionally, the invention provides peptides which contain two or moreof the same or different domain II peptides or subsequence peptidescovalently linked together.

The invention also provides peptides having an amino acid sequence whichis the amino acid sequence of human BPI from about position 142 to aboutposition 169 comprising functional domain III, having the sequence:##STR3## and subsequences thereof which have biological activity,including but not limited to one or more of the activities of BPI, forexample, bactericidal activity, LPS binding, LPS neutralization, heparinbinding or heparin neutralization. Also provided in this aspect of theinvention are peptides having substantially the same amino acid sequenceof the functional domain III peptides having the amino acid sequence ofBPI from about position 142 to about position 169 or subsequencesthereof. Additionally, the invention provides peptides which contain twoor more of the same or different domain III peptides or subsequencepeptides covalently linked together.

Also provided by this invention are interdomain combination peptides,wherein two or more peptides from different functional domains orsubsequences and variants thereof are covalently linked together.Linear, cyclized and branched-chain embodiments of these interdomaincombination peptides are provided.

The peptides of this invention have as one aspect of their utility atleast one of the known activities of BPI, including LPS binding, LPSneutralization, heparin binding, heparin neutralization and bactericidalactivity against Gram-negative bacteria. Additionally and surprisingly,some of the peptides of this invention have utility as bactericidalagents against Gram-positive bacteria. Another surprising and unexpectedutility of some of the peptides of this invention is as fungicidalagents. Peptides of this invention provide a new class of antibioticmolecules with the dual properties of neutralizing endotoxin and killingthe endotoxin-producing bacteria, useful in the treatment of mammalssuffering from diseases or conditions caused by Gram-negative bacteria.Peptides of this invention that retain this dual activity andadditionally have an increased antibiotic spectrum represent anadditional new class of antimicrobial agents. In addition, peptides ofthe invention provide a class of antimicrobial agents useful in thetreatment of infections by microbial strains that are resistant totraditional antibiotics but are sensitive to the permeability-increasingantimicrobial activity of peptides of the invention.

The invention also provides pharmaceutical compositions of the peptidesof the invention comprising the peptides or combinations of the peptidesin a pharmaceutically-acceptable carrier or diluent, both per se and foruse in methods of treating pathological or disease states or for otherappropriate therapeutic uses. Methods of using these pharmaceuticalcompositions for the treatment of pathological or disease states in amammal, including humans, are also provided by the invention. Alsoprovided by the invention are uses of BPI functional domain peptide forthe manufacture of medicaments for a variety of therapeuticapplications.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1b depict HPLC absorbance spectra for cyanogen bromide andproteolytic fragments of rBPI₂₃ ;

FIG. 2 is a graph of LAL inhibition assay results for proteolyticfragments of rBPI₂₃ ;

FIG. 3 is a graph of a heparin binding assay results using 15-mer BPIpeptides;

FIG. 4 is a graph of a Limulus Amoebocyte Lysate (LAL) inhibition assayresults using 15-mer BPI peptides;

FIG. 5 is a graph of a radial diffusion bactericidal assay results using15-mer BPI peptides;

FIG. 6 is a graph showing the effect of BPI functional domain peptidesin a heparin binding assay;

FIGS. 7a and 7b are graphs showing the effects of BPI functional domainpeptides on ATIII/heparin inhibition of thrombin;

FIGS. 8a and 8b are graphs showing the results of BPI functional domainpeptides in an LAL inhibition assay;

FIGS. 9a, 9b, 9c, and 9d are graphs showing the results of BPIfunctional domain peptides in radial diffusion bactericidal assays;

FIGS. 9e and 9f are graphs showing the results of BPI functional domainpeptides in E. coli broth assays;

FIGS. 10a, 10b, 10c, 10d and 10e are graphs showing the results of BPIfunctional domain combination peptides in radial diffusion bactericidalassays;

FIGS. 11a, 11b, 11c, 11d, 11e, 11f, 11g, 11h and 11i are graphs showingthe results of BPI functional domain peptides in radial diffusionbactericidal assays;

FIGS. 11j and 11k are graphs showing the results of BPI functionaldomain peptides in bactericidal assays on bacterial cells growing inbroth media;

FIG. 11l is a graph showing the results of BPI functional domain peptideBPI.30 in bactericidal assays performed in human serum;

FIGS. 11m and 11n are graphs showing the results of BPI functionaldomain peptides in radial diffusion bactericidal assays usingGram-positive bacteria;

FIG. 11o is a graph showing the results of BPI functional domainpeptides in radial diffusion bactericidal assays in comparison withgentamicin and vancomycin using S. aureus cells;

FIGS. 11p and 11q are graphs showing the results of BPI functionaldomain peptides in cytotoxicity assays using C. albicans cells growingin broth media;

FIGS. 12a, 12b, 12c, 12d, 12e, 12f, and 12g are graphs showing theresults of a heparin neutralization assay using BPI functional domainpeptides;

FIG. 13 is a schematic diagram of the structure of BPI domain II peptideBPI.2 (amino acid sequence 85-99 of the BPI sequence, SEQ ID NO:7);

FIG. 14 is a schematic diagram of the structure of BPI domain IIIpeptide BPI.11 (amino acid sequence 148-161 of the BPI sequence, SEQ IDNO: 13);

FIGS. 15a, 15b, 15c, 15d and 15e are graphs showing the results ofheparin binding assays using BPI functional domain substitutionpeptides;

FIG. 16 is a graph showing the results of heparin binding experimentsusing a variety of BPI functional domain peptides;

FIGS. 17a and 17b are graphs of the results of Lipid A bindingcompetition assays between synthetic BPI functional domain peptides andradiolabeled rBPI₂₃ ;

FIG. 18 is a graph of the results of Lipid A binding competition assaysbetween synthetic BPI.10 peptide and radiolabeled rBPI₂₃ in blood orphosphate buffered saline;

FIG. 19 is a graph of the results of Lipid A binding competition assaysbetween synthetic BPI peptides BPI.7, BPI.29 and BPI.30 versusradiolabeled rBPI₂₃ ;

FIGS. 20a and 20b are graphs of the results of Lipid A bindingcompetition assays between BPI functional domain peptides andradiolabeled rLBP₂₅ ;

FIG. 21 is a graph of the results of radiolabeled RaLPS bindingexperiments using BPI functional domain peptides pre-bound to HUVECcells;

FIGS. 22a, 22b, 22c, 22d, 22e, 22f, 22g, and 22h are graphs showing thevarious parameters affecting a cellular TNF cytotoxicity assay measuringthe LPS neutralization activity of BPI;

FIGS. 23a, 23b and 23c are graphs showing the dependence of NOproduction on the presence of γ-interferon and LBP in LPS-stimulated RAW264.7 cells and inhibition of such NO production using rBPI₂₃ ;

FIGS. 24a, 24b, 24c, 24d, 24e, and 24f are graphs showing LPSneutralization by BPI functional domain peptides reflected in theircapacity to inhibit NO production by RAW 264.7 cells stimulated byzymosan or LPS;

FIG. 24g is a graph showing the IC₅₀ values of synthetic BPI peptidesfor inhibition of LPS- or zymosan-stimulated NO production by RAW 264.7cells;

FIG. 25 is a schematic of rBPI₂₃ showing three functional domains;

FIG. 26a is a graph showing the dependence of LPS-mediated inhibition ofRAW 264.7 cell proliferation on the presence of rLBP;

FIGS. 26b and 26c are graphs showing patterns of BPI functional domainpeptides using the assay of Example 20D;

FIGS. 27A and 27B are graphs showing a comparison of TNF inhibition inwhole blood by various BPI functional domain peptides using the assay ofExample 20E; and

FIG. 28 is a graph showing the results of the thrombin clotting timeassay described in Example 20G using various BPI functional domainpeptides.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention provides peptides having an amino acid sequence that isthe amino acid sequence of at least one functional domain or subsequencethereof and variants of the sequence or subsequence of BPI. For thepurposes of this invention, the term "functional domain" is intended todesignate a region of the amino acid sequence of BPI that contributes tothe total biological activity of the protein. These functional domainsof BPI are defined by the activities of proteolytic cleavage fragments,overlapping 15-mer peptides and other synthetic peptides.

Domain I is defined as the amino acid sequence of BPI comprising fromabout amino acid 17 to about amino acid 45. Peptides based on thisdomain are moderately active in both the inhibition of LPS-induced LALactivity and in heparin binding assays, and do not exhibit significantbactericidal activity. Domain II is defined as the amino acid sequenceof BPI comprising from about amino acid 65 to about amino acid 99.Peptides based on this domain exhibit high LPS and heparin bindingcapacity and are bactericidal. Domain III is defined as the amino acidsequence of BPI comprising from about amino acid 142 to about amino acid169. Peptides based on this domain exhibit high LPS and heparin bindingactivity and are bactericidal.

The functional domains as herein defined include the continuous domainsI, II and III, i.e., domains comprised of a continuous portion of theBPI amino acid sequence. However, the invention also includes peptidescomprising portions of BPI which are not continuous, i.e., that areseparated in the BPI sequence. It is recognized that some non-continuousstretches of amino acid sequence may be folded in the native protein tomake such amino acid regions contiguous or in proximity, which structurecan be mimicked in the peptides of the invention by covalently linkingtogether peptides from non-continuous regions.

Peptides containing non-continuous regions of BPI amino acid sequenceare one example of combination peptides provided by the invention. Forthe purposes of this invention, combination peptides are intended toinclude linear, cyclized or branched-chain peptides comprised of two ormore peptides having an amino acid sequence from the same or differentfunctional domains of BPI and subsequences thereof. Specificallyencompassed in this definition are combinations containing from two toabout 10 functional domain peptides or subsequence thereof, preferablycombinations of two or three functional domain peptides (for example,homodimers, homotrimers, heterodimers and heterotrimers). Each of thecomponent peptides comprising such combinations may have an amino acidsequence from any particular BPI functional domain amino acid sequenceor subsequence thereof.

For purposes of this invention, the term "a biological activity of BPI"is intended to include, but is not limited to the biological activitiesof a human bactericidal/permeability-increasing protein (BPI),including, for example, a recombinant BPI holoprotein such rBPI (SEQ IDNO:69), an amino-terminal fragment of BPI such as rBPI₂₃, and mutatedamino-terminal fragments of BPI such as rBPI₂₁ Δcys (designated rBPI(1-193) ala¹³² in copending and co-assigned U.S. patent application Ser.No. 08/013,801, filed Feb. 2, 1993, incorporated by reference). Asdisclosed in copending and co-assigned U.S. patent application Ser. No.08/093,202, incorporated by reference, rBPI has been produced having thesequence set out as SEQ ID NO:69 as shown in Gray et al. (supra) exceptthat valine at position 151 is specified by GTG rather than GTC, andresidue 185 is glutamic acid (specified by GAG) rather than lysine(specified by AAG). In addition, rBPI₂₃ (see also, Gazzano-Santoro etal., 1992, Infect. Immun. 60: 4754-4761) has been produced using anexpression vector containing the 31-residue signal sequence and thefirst 199 amino acids of the sequence of rBPI with the exceptions fromthe Gray et al. (supra) sequence as noted above. Such biologicalactivities include LPS binding, LPS neutralization, heparin binding andheparin neutralization, and bactericidal activity. Specifically includedis a biological activity of any peptide of this invention that isbetween 0.1 and 10 times the activity of BPI or of a correspondingpeptide encompassing a corresponding functional domain of BPI. Alsoexpressly included in this definition of the "biological activity ofBPI" is a biological activity, for example bactericidal activity, thatis qualitatively different than the activity of BPI or the correspondingpeptide encompassing the entire corresponding domain of BPI. Forexample, such qualitative differences include differences in thespectrum of bacteria or other microorganisms against which the peptideis effective, relative to the amino acid sequence of the correspondingfunctional domain of BPI. This definition thus encompasses peptideactivities, such as bactericidal activity against Gram-positive bacteriaand fungicidal activity, not previously reported for BPI.

The invention provides peptides each of which has an amino acid sequencethat is the amino acid sequence of one of the functional domains ofhuman BPI or a subsequence thereof. Embodiments of such peptides includethe following exemplary domain I peptides single-letter abbreviationsfor amino acids can be found in G. Zubay, Biochemistry (2d. ed.), 1988(MacMillen Publishing: N.Y.), p.33!:

    __________________________________________________________________________    BPI.1 QQGTAALQKELKRIK      (SEQ ID NO: 4);                                    BPI.4 LQKELKRIKIPDYSDSFKIKHL                                                                             (SEQ ID NO: 3);                                    BPI.14                                                                              GTAALQKELKRIKIPDYSDSFKIKHLGKGH                                                                     (SEQ ID NO: 2);                                    and                                                                           BPI.54                                                                              GTAALQKELKRIKIP      (SEQ ID NO: 5);                                    the following exemplary domain II peptides:                                   BPI.2 IKISGKWKAQKRFLK      (SEQ ID NO: 7);                                    BPI.3 NVGLKFSISNANIKISGKWKAQKRFLK                                                                        (SEQ ID NO: 11);                                   and                                                                           BPI.8 KWKAQKRFLK           (SEQ ID NO: 8);                                    and the following exemplary domain III peptides:                              BPI.5 VHVHISKSKVGWLIQLFHKKIE                                                                             (SEQ ID NO: 67);                                   BPI.11                                                                              KSKVWLIQLFHKK        (SEQ ID NO: 13);                                   BPI.12                                                                              SVHVHISKSKVGWLIQLFHKKIESALRNK                                                                      (SEQ ID NO: 14);                                   BPI.13                                                                              KSKVGWLIQLFHKK       (SEQ ID NO: 15);                                   and                                                                           BPI.55                                                                              GWLIQLFHKKIESALRNKMNS                                                                              (SEQ ID NO: 61).                                   __________________________________________________________________________

It will be recognized that BPI.14, BPI.12 and BPI.55 are examples ofaddition variants.

The invention also provides linear and branched-chain combinations ofthe same or different peptides, wherein each of the peptides of thecombination has an amino acid sequence that is the amino acid sequenceof one of the functional domains of human BPI or a subsequence thereof.Embodiments of such peptides include the following exemplary combinationdomain II peptides:

    __________________________________________________________________________    BPI.9                                                                              KRFLKKWKAQKRFLK          (SEQ ID NO: 51);                                BPI.7                                                                              KWKAQKRFLKKWKAQKRFLK     (SEQ ID NO: 54);                                BPI.10.1                                                                           KRFLKKWKAQKRFLKKWKAQKRFLK                                                                              (SEQ ID NO: 55);                                and                                                                           BPI.10.2                                                                           QKRFLKKWKAQKRFLKKWKAQKRFLK                                                                             (SEQ ID NO: 65);                                and the following exemplary branched-chain domain II peptide:                 MAP. 1                                                                             (β-alanyl-Nα,Nε-substituted- Nα,Nε(B         PI.2)lysyl!lysine);                                                      and the following exemplary combination domain III peptide:                   BPI.29                                                                             KSKVGWLIQLFHKKKSKVGWLIQLFHKK                                                                           (SEQ ID NO: 56);                                and the following exemplary branched-chain domain III peptide:                MAP.2                                                                              (β-alanyl-Nα,Nε-substituted- Nα,Nε(B         PI.13)lysyl!lysine);                                                     and the following exemplary domain II-domain III interdomain combination      peptides:                                                                     BPI.30                                                                             KWKAQKRFLKKSKVGWLIQLFHKK (SEQ ID NO: 52);                                BPI.63                                                                             IKISGKWKAQKRFLKKSKVGWLIQLFHKK                                                                          (SEQ ID NO: 53);                                and                                                                           BPI.74                                                                             KSKVGWLIQLFHKKKWKAQKRFLK (SEQ ID No.: 70).                               __________________________________________________________________________

Amino acid substitution variants -are also provided, wherein the aminoacid residue at one or more positions in each of the peptides is aresidue different from the amino acid found in the correspondingposition of the BPI functional domain from which that specific peptideis derived. For example, in one embodiment of this aspect of theinvention, one position in the peptide is substituted with an alanineresidue for the amino acid found at the corresponding position in theBPI amino acid sequence. In other embodiments, one position in thepeptide is substituted with e.g., a phenylalanine, leucine, lysine ortryptophan residue for the amino acid found at the correspondingposition in the BPI amino acid sequence. Embodiments of these peptidesinclude the following exemplary substitution domain II peptides:

    ______________________________________                                        BPI.15   AKISGKWKAQKRFLK  (SEQ ID NO: 16);                                    BPI.16   IAISGKWKAQKRFLK  (SEQ ID NO: 17);                                    BPI.17   IKASGKWKAQKRFLK  (SEQ ID NO: 18);                                    BPI.18   IKIAGKWKAQKRFLK  (SEQ ID NO: 19);                                    BPI.19   IKISAKWKAQKRFLK  (SEQ ID NO: 20);                                    BPI.20   IKISGAWKAQKRFLK  (SEQ ID NO: 21);                                    BPI.21   IKISGKAKAQKRFLK  (SEQ ID NO: 22);                                    BPI.22   IKISGKWAAQKRFLK  (SEQ ID NO: 23);                                    BPI.23   IKISGKWKAAKRFLK  (SEQ ID NO: 24);                                    BPI.24   IKISGKWKAQARFLK  (SEQ ID NO: 25);                                    BPI.25   IKISGKWKAQKAFLK  (SEQ ID NO: 26);                                    BPI.26   IKISGKWKAQKRALK  (SEQ ID NO: 27);                                    BPI.27   IKISGKWKAQKRFAK  (SEQ ID NO: 28);                                    BPI.28   IKISGKWKAQKRFLA  (SEQ ID NO: 29);                                    BPI.61   IKISGKFKAQKRFLK  (SEQ ID NO: 48);                                    BPI.73   IKISGKWKAQFRFLK  (SEQ ID NO: 62);                                    BPI.77   IKISGKWKAQWRFLK  (SEQ ID NO: 72);                                    BPI.79   IKISGKWKAKKRFLK  (SEQ ID NO: 73);                                    and                                                                           BPI.81   IKISGKWKAFKRFLK  (SEQ ID NO: 75);                                    ______________________________________                                    

and the following exemplary substitution domain III peptides:

    ______________________________________                                        BPI.31   ASKVGWLIQLFHKK   (SEQ ID NO: 33);                                    BPI.32   KAKVGWLIQLFHKK   (SEQ ID NO: 34);                                    BPI.33   KSAVGWLIQLFHKK   (SEQ ID NO: 35);                                    BPI.34   KSKAGWLIQLFHKK   (SEQ ID NO: 36);                                    BPI.35   KSKVAWLIQLFHKK   (SEQ ID NO: 37);                                    BPI.36   KSKVGALIQLFHKK   (SEQ ID NO: 38);                                    BPI.37   KSKVGWAIQLFHKK   (SEQ ID NO: 39);                                    BPI.38   KSKVGWLAQLFHKK   (SEQ ID NO: 40);                                    BPI.39   KSKVGWLIALFHKK   (SEQ ID NO: 41);                                    BPI.40   KSKVGWLIQAFHKK   (SEQ ID NO: 42);                                    BPI.41   KSKVGWLIQLAHKK   (SEQ ID NO: 43);                                    BPI.42   KSKVGWLIQLFAKK   (SEQ ID NO: 44);                                    BPI.43   KSKVGWLIQLFHAK   (SEQ ID NO: 45);                                    BPI.44   KSKVGWLIQLFHKA   (SEQ ID NO: 46);                                    BPI.82   KSKVGWLIQLWHKK   (SEQ ID NO: 76);                                    BPI.85   KSKVLWLIQLFHKK   (SEQ ID NO: 79);                                    BPI.86   KSKVGWLILLFHKK   (SEQ ID NO: 80);                                    BPI.87   KSKVGWLIQLFLKK   (SEQ ID NO: 81);                                    BPI.91   KSKVGWLIFLFHKK   (SEQ ID NO: 86);                                    BPI.92   KSKVGWLIKLFHKK   (SEQ ID NO: 87);                                    BPI.94   KSKVGWLIQLFFKK   (SEQ ID NO: 89);                                    BPI.95   KSKVFWLIQLFHKK   (SEQ ID NO: 90);                                    BPI.96   KSKVGWLIQLFHKF   (SEQ ID NO: 91);                                    and                                                                           BPI.97   KSKVKWLIQLFHKK   (SEQ ID NO: 92).                                    ______________________________________                                    

A particular utility of such single amino acid-substituted BPIfunctional domain peptides provided by the invention is to identifycritical residues in the peptide sequence, whereby substitution of theresidue at a particular position in the amino acid sequence has adetectable effect on at least one of the biological activities of thepeptide. Expressly encompassed within the scope of this invention areembodiments of the peptides of the invention having substitutions atsuch critical residues so identified using any amino acid, whethernaturally-occurring or atypical, wherein the resulting substitutedpeptide has biological activity as defined herein.

Substituted peptides are also provided that are multiple substitutions,i.e., where two or more different amino acid residues in the functionaldomain amino acid sequence are each substituted with another amino acid.For example, in embodiments of such doubly-substituted peptides, bothpositions in the peptide are substituted e.g., with alanine,phenylalanine or lysine residues for the amino acid found at thecorresponding positions in the BPI amino acid sequence. Examples ofembodiments of these peptides include the multiply substituted domain IIpeptides:

    ______________________________________                                        BPI.45   IKISGKWKAAARFLK  (SEQ ID NO: 31);                                    BPI.56   IKISGKWKAKQRFLK  (SEQ ID NO: 47);                                    BPI.59   IKISGAWAAQKRFLK  (SEQ ID NO: 30);                                    BPI.60   IAISGKWKAQKRFLA  (SEQ ID NO: 32);                                    and                                                                           BPI.88   IKISGKWKAFFRFLK  (SEQ ID NO: 82);                                    ______________________________________                                    

and the exemplary multiply substituted domain III peptide:

    ______________________________________                                        BPI.100   KSKVKWLIKLFHKK  (SEQ ID NO: 94);                                    ______________________________________                                    

and the following exemplary multiply substituted domain II substitutioncombination peptide:

    __________________________________________________________________________    BPI.101                                                                              KSKVKWLIKLFFKFKSKVKWLIKLFFKF                                                                      (SEQ ID NO: 95);                                   __________________________________________________________________________

and the following exemplary multiply substituted domain II-domain IIIinterdomain substitution combination peptide:

    ______________________________________                                        BPI.102                                                                              KWKAQFRFLKKSKVGWLILLFHKK                                                                           (SEQ ID NO: 96).                                  ______________________________________                                    

Another aspect of such amino acid substitution variants are those wherethe substituted amino acid residue is an atypical amino acid.Specifically encompassed in this aspect of the peptides of the inventionare peptides containing D-amino acids, modified ornon-naturally-occurring amino acids, and altered amino acids to providepeptides with increased stability, potency or bioavailability.Embodiments of these peptides include the following exemplary domain IIpeptides with atypical amino acids:

    ______________________________________                                        BPI.66 IKISGKW.sub.D KAQKRFLK                                                                            (SEQ ID NO: 49);                                   BPI.67 IKISGKA.sub.β-(1-naphthyl) KAQKRFLK                                                          (SEQ ID NO: 50);                                   BPI.70 IKISGKA.sub.β-(3-pyridyl) KAQKRFLK                                                           (SEQ ID NO: 63);                                   BPI.71 A.sub.D A.sub.D IKISGKWKAQKRFLK                                                                   (SEQ ID NO: 66);                                   BPI.72 IKISGKWKAQKRA.sub.β-(3-pyridyl) LK                                                           (SEQ ID NO: 64);                                   BPI.76 IKISGKWKAQF.sub.D RFLK                                                                            (SEQ ID NO: 71);                                   BPI.80 IKISGKWKAQA.sub.β-(1-naphthyl) RFLK                                                          (SEQ ID NO: 74);                                   BPI.84 IKISGKA.sub.β-(1-naphthyl) KAQFRFLK                                                          (SEQ ID NO: 78);                                   BPI.89 IKISGKA.sub.β-(1-naphthyl) KAFKRFLK                                                          (SEQ ID NO: 84);                                   and                                                                           BPI.90 IKISGKA.sub.β-(1-naphthyl) KAFFRFLK                                                          (SEQ ID NO: 85);                                   ______________________________________                                    

the exemplary domain III peptide with atypical amino acids:

    ______________________________________                                        BPI.83 KSKVGA.sub.β-(1-naphthyl) LIQLFHKK                                                           (SEQ ID NO: 77);                                   ______________________________________                                    

and the exemplary domain II-domain III interdomain combination peptideswith atypical amino acids:

    __________________________________________________________________________    BPI.93                                                                            IKISGKA.sub.β-(1-naphthyl) KAQFRFLKKSKVGWLIQLFHKK                                                (SEQ ID NO: 88);                                  and                                                                           BPI.98                                                                            IKISGKA.sub.β-(1-naphthyl) KAQFRFLKKSKVGWLIFLFHKK                                                (SEQ ID NO: 83).                                  __________________________________________________________________________

Linear and branched-chain combination embodiments of the amino acidsubstitution variant peptides, which create multiple substitutions inmultiple domains, are also an aspect of this invention. Embodiments ofthese peptides include the following exemplary combination/substitutiondomain II peptides:

    __________________________________________________________________________    BPI.46                                                                             KWKAAARFLKKWKAQRFLK    (SEQ ID NO: 57);                                  BPI.47                                                                             KWKAQKRFLKKWKAAARFLK   (SEQ ID NO: 58);                                  BPI.48                                                                             KWKAAARFLKKWAAAKRFLK   (SEQ ID NO: 59);                                  BPI.69                                                                             KWKAAARFLKKWKAAARFLKKWKAAARFLK                                                                       (SEQ ID NO: 60);                                  and                                                                           BPI.99                                                                             KWKAQWRFLKKWKAQWRFLKKWKAQWRFLK                                                                       (SEQ ID NO: 93).                                  __________________________________________________________________________

Dimerized and cyclized embodiments of each of the aforementioned BPIfunctional domain peptides are also provided by this invention.Embodiments of these peptides include the following exemplarycysteine-modified domain II peptides:

    ______________________________________                                        BPI.58    CIKISGKWKAQKRFLK (SEQ ID NO: 9);                                    BPI.65 (red)                                                                            CIKISGKWKAQKRFLKC                                                                              (SEQ ID NO: 68);                                   and       S - S                                                               BPI.65 (ox.)                                                                            CIKISGKWKAQKRFLKC                                                                              (SEQ ID NO: 10).                                   ______________________________________                                    

BPI functional domain peptides described herein are useful as potentanti-bacterial agents for Gram-negative bacteria and for neutralizingthe adverse effects of LPS associated with the cell membranes ofGram-negative bacteria. The peptides of the invention have, in varyingamounts, additional activities of BPI, including activities not directlyassociated with the Gram-negative bacterial infection, such as heparinbinding and neutralization. Peptides provided by this invention also mayhave biological activities distinct from the known biological activitiesof BPI. For example, some embodiments of the peptides of the inventionsurprisingly have been found to have a biological target range forbactericidal activity that is broader than BPI and exhibits bactericidalactivity against Gram-positive as well as Gram-negative bacteria. Someembodiments of the invention have surprisingly been found to havefungicidal activity. Thus, the invention advantageously providespeptides having amino acid sequences of the biologically functionaldomains of BPI having distinct antimicrobial activities. Peptides ofthis invention that possess the dual anti-bacterial and anti-endotoxicproperties of BPI, including those with an increased antibioticspectrum, represent a new class of antibiotic molecules.

BPI functional domain peptides of the invention will have biologicaltherapeutic utilities heretofor recognized for BPI protein products. Forexample, co-owned, copending U.S. patent application Ser. No. 08/188,221filed Jan. 24, 1994, addresses use of BPI protein products in thetreatment of humans exposed to Gram-negative bacterial endotoxin incirculation. Co-owned, copending U.S. patent application Ser. No.08/031,145 filed Mar. 12, 1993 addresses administration of BPI proteinproducts for treatment of mycobacterial diseases. Co-owned, copendingU.S. patent apatent application Ser. No. 08/132,510, filed Oct. 5, 1993,addresses use of BPI protein products in the treatment of conditionsinvolving depressed reticuloendothelial system function. Co-owned,copending U.S. patent application Ser. No. 08/125,651, filed Sep. 22,1993, addresses synergistic combinations of BPI protein products andantibiotics. Co-owned, copending U.S. patent application Ser. No.08/093,201 filed Jul. 14, 1993, addresses methods of potentiating BPIprotein product bactericidal activity by administration of LBP proteinproducts. Co-owned, copending U.S. patent application Ser. No.08/031,144 filed Mar. 12, 1993, addresses administration of BPI proteinproducts for treatment of Helicobacterial infections. The disclosures ofthe above applications are specifically incorporated by reference hereinfor the purpose of exemplifying therapeutic uses for BPI functionaldomain peptides of the invention. The BPI functional domain peptides ofthe invention also have therapeutic utility for the treatment ofpathological conditions and disease states as disclosed in the aboveidentified U.S. patent application Ser. Nos. 08/030,644, 08/093,202 and08/183,222 parent applications.

BPI functional domain peptides of the invention are thus useful inmethods for: neutralizing the anti coagulant effect of heparin;inhibiting angiogenesis (especially angiogenesis associated with ocularretinopathy); inhibiting endothelial cell proliferation (especiallyendometriosis and proliferation associated with implantation offertilized ova); inhibiting malignant tumor cell proliferation(especially Kaposi's sarcoma proliferation); treating chronicinflammatory disease states (such as arthritis and especially reactiveand rheumatoid arthritis); treating Gram-negative bacterial infectionand the sequelae thereof; treating the adverse effects (such asincreased cytokine production) of Gram-negative endotoxin in bloodcirculation; killing Gram-negative bacteria; treating adversephysiological effects associated with depressed reticuloendothelialsystem function (especially involving depressed function of Kupffercells of the liver such as results from physical, chemical andbiological insult to the liver); treating, in synergistic combinationwith antibiotics (such as gentamicin, polymycin B and cefamandolenafate) Gram-negative bacterial infection and the sequelae thereof;killing Gram-negative bacteria in synergistic combination withantibiotics; treating, in combination with LBP protein products,Gram-negative bacterial infection and the sequelae thereof; killingGram-negative bacteria in combination with LBP protein products;treating, alone or in combination with antibiotics and/or bismuth,Mycobacteria infection (especially infection by M. tuberculosis, M.leprae and M. avium); treating adverse physiological effects (such asincreased cytokine production) of lipoarabinomannan in bloodcirculation; decontaminating fluids (such as blood, plasma, serum andbone marrow) containing lipoarabinomannan; and, treating disease states(such as gastritis and peptic, gastric and duodenal ulcers) associatedwith infection by bacteria of the genus Helicobacter. The presentinvention also provides pharmaceutical compositions for oral,parenteral, topical and aerosol administration comprising BPI functionaldomain peptides in amounts effective for the uses noted above andespecially compositions additionally comprising pharmaceuticallyacceptable diluents, adjuvants or carriers.

With respect to uses of BPI functional domain peptides in combinationwith LBP protein products, as used herein, "LBP protein product"includes naturally and recombinantly product lipopolysaccharide bindingprotein; natural, synthetic, and recombinant biologically activepolypeptide fragments and derivatives of lipopolysaccharide bindingprotein; and biologically active polypeptide analogs, including hybridfusion proteins, of either LBP or biologically active fragments thereof.LBP protein products useful according to the methods of the presentinvention include LBP holoprotein which can be produced by expression ofrecombinant genes in transformed eucaryotic host cells such as describedin co-owned and copending U.S. patent application Ser. No. 08/079,510filed Jun. 17, 1993 and designated rLBP. Also described in thatapplication are preferred LBP protein derivatives which lackCD14-mediated inflammatory properties and particularly the ability tomediate LPS activity through the CD14 receptor. Such LBP proteinproducts are preferred for use according to the present inventionbecause excessive CD14-mediated immunostimulation is generallyconsidered undesirable, and is particularly so in subjects sufferingfrom infection.

Preferred LBP protein derivatives are characterized as amino-terminalfragments having a molecular weight of about 25 kD. Most preferred areLBP amino-terminal fragments characterized by the amino acid sequence ofthe first 197 amino acids of the amino-terminus of LBP, as set out inSEQ ID NOS:97 and 98, designated rLBP₂₅, the production of which isdescribed in previously-noted co-owned and copending U.S. patentapplication Ser. No. 08/079,510. It is contemplated that LBP proteinderivatives considerably smaller than 25 kD and comprising substantiallyfewer than the first 197 amino acids of the amino-terminus of theholo-LBP molecule are suitable for use according to the inventionprovided they retain the ability to bind to LPS. Moreover, it iscontemplated that LBP protein derivatives comprising greater than thefirst 197 amino acid residues of the holo-LBP molecule including aminoacids on the carboxy-terminal side of first 197 amino acids of the rLBPas disclosed in SEQ ID NOS: 97 and 98 will likewise prove usefulaccording to the methods of the invention provided they lack an elementthat promotes CD 14-mediated immunostimulatory activity. It is furthercontemplated that those of skill in the art are capable of makingadditions, deletions and substitutions of the amino acid residues of SEQID NOS: 97 and 98 without loss of the desired biological activities ofthe molecules. Still further, LBP protein products may be obtained bydeletion, substitution, addition or mutation, including mutation bysite-directed mutagenesis of the DNA sequence encoding the LBPholoprotein, wherein the LBP protein product maintains LPS-bindingactivity and-lacks CD14-mediated immunostimulatory activity.Specifically contemplated are LBP hybrid molecules and dimeric formswhich may result in improved affinity of LBP for bacteria and/orincreased stability in vivo. These include LBP/BPI hybrid proteins andLBP-Ig fusion proteins. Such hybrid proteins further include those usinghuman gamma 1 or gamma 3 hinge regions to permit dimer formation. Otherforms of dimer contemplated to have enhanced serum stability and bindingaffinity include fusions with Fc lacking the CH₂ domain, or hybridsusing leucine or helix bundles.

BPI functional domain peptides of the invention may be generated and/orisolated by any means known in the art, including by means ofrecombinant production. Co-owned U.S. Pat. No. 5,028,530, issued Jul. 2,1991, co-owned U.S. Pat. No. 5,206,154, issued Apr. 27, 1993, andco-owned, copending U.S. patent application Ser. No. 08/010,676, filedJan. 28, 1993, all of which are hereby incorporated by reference,disclose novel methods for the recombinant production of polypeptides,including antimicrobial peptides. Additional procedures for recombinantproduction of antimicrobial peptides in bacteria have been described byPiers et al., 1993, Gene 134: 7-13. Co-owned, copending U.S. patentapplication Ser. No. 07/885,501, filed May 19, 1992, and acontinuation-in-part thereof, U.S. patent application Ser. No.08/072,063, filed May 19, 1993 which are both hereby incorporated byreference, disclose novel methods for the purification of recombinantBPI expressed in and secreted from genetically transformed mammalianhost cells in culture and discloses how one may produce large quantitiesof recombinant BPI suitable for incorporation into stable, homogeneouspharmaceutical preparations.

BPI functional domain peptides may also be advantageously produced usingany such methods. Those of ordinary skill in the art are able to isolateor chemically synthesize a nucleic acid encoding each of the peptides ofthe invention. Such nucleic acids are advantageously utilized ascomponents of recombinant expression constructs, wherein the nucleicacids are operably linked with transcriptional and/or translationalcontrol elements, whereby such recombinant expression constructs arecapable of expressing the peptides of the invention in cultures ofprokaryotic, or preferably eukaryotic cells, most preferably mammaliancells, transformed with such recombinant expression constructs.

Peptides of the invention may be advantageously synthesized by any ofthe chemical synthesis techniques known in the art, particularlysolid-phase synthesis techniques, for example, usingcommercially-available automated peptide synthesizers. Such peptides mayalso be provided in the form of combination peptides, wherein thepeptides comprising the combination are linked in a linear fashion oneto another and wherein a BPI sequence is present repeatedly in thepeptide, with or without separation by "spacer" amino acids allowing forselected conformational presentation. Also provided are branched-chaincombinations, wherein the component peptides are covalently linked viafunctionalities in amino acid sidechains of the amino acids comprisingthe peptides.

Functional domain peptides of this invention can be provided asrecombinant hybrid fusion proteins comprising BPI functional domainpeptides and at least a portion of at least one other polypeptide. Suchproteins are described, for example, by Theofan et al. in co-owned,copending U.S. patent application Ser. No. 07/885,911, filed May 19,1992, and a continuation-in-part application thereof, U.S. patentapplication Ser. No. 08/064,693, filed May 19, 1993, which areincorporated herein by reference in their entirety.

Generally, those skilled in the art will recognize that peptides asdescribed herein may be modified by a variety of chemical techniques toproduce compounds having essentially the same activity as the unmodifiedpeptide, and optionally having other desirable properties. For example,carboxylic acid groups of the peptide, whether carboxyl-terminal orsidechain, may be provided in the form of a salt of apharmaceutically-acceptable cation or esterified to form a C₁ -C₁₆ester, or converted to an amide of formula NR₁ R₂ wherein R₁ and R₂ areeach independently H or C₁ -C₁₆ alkyl, or combined to form aheterocyclic ring, such as 5- or 6-membered. Amino groups of thepeptide, whether amino-terminal or sidechain, may be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C₁ -C₁₆ alkyl or dialkyl amino or furtherconverted to an amide. Hydroxyl groups of the peptide sidechain may beconverted to C₁ -C₁₆ alkoxy or to a C₁ -C₁₆ ester using well-recognizedtechniques. Phenyl and phenolic rings of the peptide sidechain may besubstituted with one or more halogen atoms, such as fluorine, chlorine,bromine or iodine, or with C₁ -C₁₆ alkyl, C₁ -C₁₆ alkoxy, carboxylicacids and esters thereof, or amides of such carboxylic acids. Methylenegroups of the peptide sidechains can be extended to homologous C₂ -C₄alkylenes. Thiols can be protected with any one of a number ofwell-recognized protecting groups, such as acetamide groups. Thoseskilled in the art will also recognize methods for introducing cyclicstructures into the peptides of this invention to select and provideconformational constraints to the structure that result in enhancedbinding and/or stability. For example, a carboxyl-terminal oramino-terminal cysteine residue can be added to the peptide, so thatwhen oxidized the peptide will contain a disulfide bond, therebygenerating a cyclic peptide. Other peptide cyclizing methods include theformation of thioethers and carboxyl- and amino-terminal amides andesters.

Peptidomimetic and organomimetic embodiments are also hereby explicitlydeclared to be within the scope of the present invention, whereby thethree-dimensional arrangement of the chemical constituents of suchpeptido- and organomimetics mimic the three-dimensional arrangement ofthe peptide backbone and component amino acid sidechains in the peptide,resulting in such peptido- and organomimetics of the peptides of thisinvention having substantial biological activity. It is implied that apharmacophore exists for each of the described activities of BPI. Apharmacophore is an idealized, three-dimensional definition of thestructural requirements for biological activity. Peptido- andorganomimetics can be designed to fit each pharmacophore with currentcomputer modelling software (computer aided drug design). The degree ofoverlap between the specific activities of pharmacophores remains to bedetermined.

The administration of BPI functional domain peptides is preferablyaccomplished with a pharmaceutical composition comprising a BPIfunctional domain peptide and a pharmaceutically acceptable diluent,adjuvant, or carrier. The BPI functional domain peptide composition maybe administered without or in conjunction with known antibiotics,surfactants, or other chemotherapeutic agents. Examples of suchcombinations are described in co-owned, copending, U.S. patentapplication Ser. No. 08/012,360, filed Feb. 2, 1993, andcontinuation-in-part U.S. patent application Ser. No. 08/190,869, filedFeb. 2, 1994, the disclosures of which are incorporated herein byreference.

Effective doses of BPI functional domain peptides for bactericidalactivity, partial or complete neutralization of the anti-coagulantactivity of heparin, partial or complete neutralization of LPS and othereffects described herein may be readily determined by those of skill inthe art according to conventional parameters, each associated with thecorresponding biological activity, including, for example, the size ofthe subject, the extent and nature of the bacterial infection, theextent and nature of the endotoxic shock, and the quantity of heparinadministered to the subject and the time since administration of theheparin. Similar determinations will be made by those of skill in thisart for using the peptide embodiments of this invention for therapeuticuses envisioned and described herein.

Embodiments of the invention comprising medicaments can be prepared fororal administration, for injection, or other parenteral methods andpreferably include conventional pharmaceutically acceptable carriers,adjuvents and counterions as would be known to those of skill in theart. The medicaments are preferably in the form of a unit dose in solid,semi-solid and liquid dosage forms such as tablets, pills, powders,liquid solutions or suspensions, and injectable and infusible solutions.Effective dosage ranges from about 100 μg/kg to about 10 mg/kg of bodyweight are contemplated.

The Examples which follow are illustrative of specific embodiments ofthe invention, and various uses thereof. Example 1 describes thepreparation of proteolytic fragments of BPI; Example 2 describes theresults of bactericidal assays of the proteolytic fragments of Example1; Example 3 describes the results of heparin binding assays using theproteolytic fragments of Example 1; Example 4 describes the results ofexperiments using Limulus amebocyte lysates to assay the LPS bindingactivity of the proteolytic fragments of Example 1; Example 5 describesthe preparation of 15-mer peptides of BPI; Example 6 describes theresults of heparin binding assays using the 15-mer peptides of Example5; Example 7 describes the results of Limulus amebocyte lysates assaysusing the 15-mer peptides of Example 5; Example 8 describes the resultsof bactericidal assays of the 15-mer peptides of Example 5; Example 9describes the preparation of BPI individual functional domain peptides;Example 10 describes the results of heparin binding assays using the BPIindividual functional domain peptides of Example 9; Example 11 describesthe results of heparin neutralization assays using the BPI individualfunctional domain peptides of Example 9; Example 12 describes theresults of Limulus amebocyte lysates assays of LPS neutralizationactivity using the BPI individual functional domain peptides of Example9; Example 13 describes the results of bactericidal assays of the BPIindividual functional domain peptides of Example 9; Example 14 describesthe preparation of BPI combination functional domain peptides; Example15 describes the results of bactericidal activity assays of the BPIcombination functional domain peptides of Example 14; Example 16describes the results of additional bactericidal activity assays of theBPI combination functional domain peptides of Example 14; Example 17describes the results of in vivo and in vitro heparin neutralizationassays using the BPI combination functional domain peptides of Example14; Example 18 describes the preparation and functional activityanalysis of bactericidal activity, heparin binding activity and LPSneutralization activity assays of BPI substitution variant functionaldomain peptides; Example 19 provides a summary of the results ofbactericidal and heparin binding assays using representative BPIfunctional domain peptides; Example 20 describes analysis of BPIfunctional domain peptides in a variety of binding and neutralizationassays; Example 21 addresses a heparin neutralization assay; Example 22describes administration of BPI functional domain peptides in modelsystems of collagen and bacteria-induced arthritis animal model systemsexemplifying treatment of chronic inflammatory disease states; Example23 illustrates testing of BPI functional domain peptides for angiostaticeffects in a mouse malignant melanoma metastasis model system; Example24 addresses effects of BPI functional domain peptides on endothelialcell proliferation; Example 25 describes analysis of BPI functionaldomain peptides in animal model systems; and Example 26 describes aprotocol for testing the anti-endotoxin effects of BPI functional domainpeptides of the invention in vivo in humans.

EXAMPLE 1 Preparation of BPI Proteolytic Fragments

Chemical cleavage and enzymatic digestion processes were applied torBPI₂₃ to produce variously-sized proteolytic fragments of therecombinant BPI protein.

rBPI₂₃ protein was reduced and alkylated prior to proteolysis bycyanogen bromide (CNBr) or endoproteinase Asp-N. The protein wasdesalted by overnight precipitation upon the addition of cold (4° C.)acetone (1:1 v/v) and the precipitated protein recovered by pelletingunder centrifugation (5000 ×g) for 10 minutes. The rBPI₂₃ protein pelletwas washed twice with cold acetone and dried under a stream of nitrogen.An rBPI₂₃ solution was then reconstituted to a final concentration of 1mg protein/mL in 8M urea/0.1M Tris-HCl (pH 8.1) and reduced by additionof 3.4 mM dithiothreitol (Calbiochem, San Diego, Calif.) for 90 minutesat 37° C. Alkylation was performed by the addition of iodoacetamide(Sigma Chemical Co., St. Louis, Mo.) to a final concentration of 5.3millimolar and incubation for 30 minutes in the dark at roomtemperature. The reduced and alkylated protein was acetone-precipitated,centrifuged and washed as described above and the pellet was redissolvedas described below for either CNBr or Asp-N digestion.

For CNBr-catalyzed protein fragmentation, the washed pellet was firstdissolved in 70% trifluoroacetic acid (TFA) (Protein Sequencing Grade,Sigma Chemical Co., St. Louis, Mo.) to a final protein concentration of5 mg/mL. Cyanogen bromide (Baker Analyzed Reagent, VWR Scientific, SanFrancisco, Calif.) dissolved in 70% TFA was added to give a final ratioof 2:1 CNBr to protein (w/w). This ratio resulted in an approximately75-fold molar excess of CNBr relative to the number of methionineresidues in the rBPI₂₃ protein. The reaction was purged with nitrogenand allowed to proceed for 24 hours in the dark at room temperature. Thereaction was terminated by adding 9 volumes of distilled water, andfollowed by freezing (-70° C.) and lyophilization.

For endoproteinase digestion, the reduced and alkylated rBPI₂₃ wassolubilized at a concentration of 5.0 mg/mL in 8M urea/0.1M Tris-HCl (pH8.1). An equal volume of 0.1M Tris-HCl (pH 8.1) was then added so thatthe final conditions were 2.5 mg/mL protein in 5M urea/0.1M Tris-HCl (pH8.1). Endoproteinase Asp-N from Pseudomonas fragi (Boehringer-Mannheim,Indianapolis, Ind.) was added at a 1:1000 (w/w, enzyme:substrate) ratio,and digestion was allowed to proceed for 6 hours at 37° C. The reactionwas terminated by addition of TFA to a final concentration of 0.1% andthe samples were then fractionated by reverse phase HPLC.

The CNBr and Asp-N fragment mixtures were purified on a Zorbax ProteinPlus C₃ column (4.6×250 mm, 300 Å pore size, MACMOD Analytical Inc,Chadsford, Pa.). A gradient ranging from 5% acetonitrile in 0.1% TFA to80% acetonitrile in 0.1 % TFA was run over this column over a 2 hourelution period at a flow rate of 1.0 mL/min. Fragment elution wasmonitored at 220 nm using a Beckman System Gold HPLC (Beckman ScientificInstruments, San Ramon, Calif.). The column heating compartment wasmaintained at 35° C. and the fractions were collected manually, frozenat -70° C. and dried in a Speed Vac concentrator. Fragments were thensolubilized in a solution of 20 mM sodium acetate (pH 4.0)/0.5M NaClprior to use.

Electrospray ionization mass spectrometry (ESI-MS) was performed on a VGBio-Q mass spectrometer by Dr. Francis Bitsch and Mr. John Kim in thelaboratory of Dr. Cedric Shackleton, Children's Hospital-OaklandResearch Institute. Molecular masses were obtained by mathematicaltransformation of the data.

Although the DNA sequence for rBPI₂₃ encodes amino acid residues 1-199of the mature protein, a significant portion of the protein that isproduced is truncated at Leu-193 and Val-195, as determined by ESI-MS.The existence of these carboxyl-terminal truncations were verified byisolating the carboxyl-terminal tryptic peptides, which were sequencedand analyzed by ESI-MS.

There are six methionine residues in the rBPI₂₃ protein, at positions56, 70, 100, 111, 170, and 196, and chemical cleavage by cyanogenbromide produced six major peptide fragments as predicted. The resultsof the CNBr cleavage experiments are summarized in Table I. Thefragments were isolated by reverse phase (C₃) HPLC (FIG. 1a) and theiramino-terminal sequences were determined by Edman degradation. The twolargest fragments (C1 and C5) were not resolved by the C₃ HPLC columnand further attempts to resolve them by ion exchange chromatography wereunsuccessful, presumably because they are similar in length andisoelectric point. The identities of the C1, C5 fragments within themixture were determined by ESI-MS. The predicted mass of C1 is 6269(Table I), taking into account the loss of 30 a.m.u. resulting from theconversion of the carboxyl-terminal methionine to homoserine during theCNBr cleavage reaction. The observed mass of 6251.51±0.34 is consistentwith the loss of a water molecule (18 a.m.u.) in a homoserine lactoneintermediate, which may be favored over the formation of the homoserinebecause of the hydrophobicity of the C1 fragment C-terminal amino acids.The predicted mass of the C5 fragment is 6487 and the observed mass is6385.84±0.39 (Table I). For the C5 fragment, the C-terminal amino acidsare hydrophilic, so the hydrolysis of the homoserine lactoneintermediate is probably favored. From both the amino-terminalsequencing and the mass spectrum data, the C5 component representsapproximately 10-25% of the material in the C1/C5 mixture.

Proteolytic cleavage with endoproteinase Asp-N was performed to provideadditional fragments for the regions contained within the CNBr C1/C5mixture. There are six aspartic acid residues within the rBPI₂₃ sequenceat positions 15, 36, 39, 57, 105, and 116. The six major Asp-N fragmentsisolated by C₃ HPLC (FIG. 1b) were sequenced and masses were determinedby ESI-MS (Table I). A short duration digest at a 1:1000 (w/w,enzyme:substrate) ratio was used to eliminate potential non-specificcleavages, particularly at glutamic acid residues. It is evident thatthis digestion did not continue until completion, as one fragment (1-38)was isolated where Asp residues (amino acids 15 and 35) were notcleaved. The mass spectra of the Asp-N fragments were consistent withthe predicted masses for each individual fragment. Unlike the CNBrcleavage, where the carboxyl-terminal fragment was poorly resolved, theAsp-N fragment from amino acid 116 to the carboxyl-terminus was wellresolved from all of the other Asp-N fragments.

                  TABLE I                                                         ______________________________________                                        Summary of rBPI.sub.23 Cleavage Fragment Analysis                                                MASS                                                       PEAK   SEQUENCE   I.D.       measured                                                                              predicted                                ______________________________________                                        CNBr Cleavage Fragments                                                       I      101-110    C4 (101-111)                                                                             N.D.    1169                                     II     57-67      C2 (57-70) N.D.    1651                                     III    71-99      C3 (71-100)                                                                              N.D.    3404                                     IV     171-194    C6 (171-196)                                                                             N.D.    2929                                     V      1-25, 112-124                                                                            C1 (1-56), 6251    6269                                                       C5 (112-170)                                                                             6486    6487                                     Asp--N Proteolytic Fragments                                                  A       1-14      A1 (1-14)  1465.5  1464                                     I      39-56      A3 (39-56) 2145.2  2145                                     II     15-38      A2 (15-38) 2723.6  2724                                     III    57-76      A4 (57-104)                                                                              5442.5  5442                                     IV      1-38      A1 A2 (1-38)                                                                             4171.4  4172                                     VI     116-134    A6a (116-193)                                                                            8800.3  8800                                     VII    116-128    A6b (116-195)                                                                            8997.1  8996                                     ______________________________________                                    

EXAMPLE 2 Bactericidal Effects of BPI Proteolytic Fragments

BPI proteolytic fragments produced according to Example 1 were screenedfor bactericidal effects using rough mutant E. coli J5 bacteria in aradial diffusion assay. Specifically, an overnight culture of E. coli 35was diluted 1:50 into fresh tryptic soy broth and incubated for 3 hoursat 37° C. to attain log phase growth of the culture. Bacteria were thenpelleted at 3,000 rpm for 5 minutes in a Sorvall RT6000B centrifuge(Sorvall Instruments, Newton, Conn.). 5 mL of 10 mM sodium phosphatebuffer (pH 7.4) was added and the preparation was re-pelleted. Thesupernatant was decanted and 5 mL of fresh buffer was added, thebacteria were resuspended and their concentration was determined bymeasurement of absorbance at 590 nm (an Absorbance value of 1.00 at thiswavelength equals a concentration of 1.25×10⁹ CFU/mL in suspension). Thebacteria were diluted to 4×10⁶ CFU/mL in 10 mL of molten underlayeragarose (at approximately 45° C.) and inverted repeatedly to mix in 15mL polypropylene tubes conventionally used for this purpose.

The entire contents of such tubes were then poured into a level squarepetri dish and distributed evenly by rocking the dish side-to-side. Theagarose hardened in less than 30 seconds and had a uniform thickness ofabout 1 mm. A series of wells were then punched into the hardenedagarose using a sterile 3 mm punch attached to a vacuum apparatus. Thepunch was sterilized with 100% alcohol and allowed to air dry prior touse to avoid contaminating the bacterial culture.

5 or 10 μL of each of the BPI fragments were carefully pipetted intoeach well. As a negative control, dilution buffer (pH 8.3) was added toa separate well, and rBPI₂₃ at concentrations of 5 μg/mL and 1 μg/mLwere also added as positive controls. Each plate was incubated at 37° C.for 3 hours, and then 10 mL of molten overlayer agarose (atapproximately 45° C.) was added into the level petri dish, allowed toharden and incubated overnight at 37° C. The next day, a clear zone wasseen against the lawn of bacteria in those wells having bactericidalactivity. In order to visually enhance this zone, a dilute Coomassiesolution (consisting of 0.002% Coomassie Brilliant Blue, 27% methanol,15% formaldehyde (37% stock solution) and water) was poured over theagar and allowed to stain for 24 hours. The bacterial zones weremeasured with a micrometer.

No bactericidal activity was discerned for the rBPI₂₃ fragmentsgenerated by CNBr or by Asp-N digestion, when tested at amounts up to 25pmol/well. In contrast, this assay detected measurable bactericidalactivity using rBPI₂₃ in amounts as low as 0.75 pmol/well. Reduced andalkylated rBPI₂₃, on the other hand, also was not bactericidal atamounts up to 100 pmol/well, while alkylated rBPI₂₃ retainedbactericidal activity equivalent to rBPI₂₃.

EXAMPLE 3 Heparin Binding by BPI Proteolytic Fragments

rBPI₂₃ and the BPI proteolytic fragments produced according to Example 1were evaluated in heparin binding assays according to the methodsdescribed in Example 1 in copending U.S. patent application Ser. No.08/093,202, filed Jul. 15, 1993 and incorporated by reference. Briefly,each fragment was added to wells of a 96-well microtiter plate having apolyvinylidene difluoride membrane (Immobilon-P, Millipore, Bedford,Mass.) disposed at the bottom of the wells. Heparin binding of CNBrfragments was estimated using 100 picomoles of each fragment per wellwith a saturating concentration of ³ H-heparin (20 μg/mL). Positivecontrol wells contained varying amounts of rBPI₂₃. The wells were driedand subsequently blocked with a 0.1% bovine serum albumin (BSA) inphosphate buffered saline, pH 7.4 (blocking buffer). Dilutions of ³H-heparin (0.03-20 μCi/ml, avg. M.W.=15,000; DuPont-NEN, Wilmington,Del.) were made in the blocking buffer and incubated in the BPIpeptide-containing wells for one hour at 4° C. The unbound heparin wasaspirated and the wells were washed three times with blocking buffer,dried and removed for quantitation in a liquid scintillation counter(Model 1217, LKB, Gaithersburg, Md.). Although BSA in the blockingbuffer did show a low affinity and capacity to bind heparin, this wasconsidered physiologically irrelevant and the background was routinelysubtracted from the test compound signal. The specificity offragment-heparin binding was established by showing that the binding ofradiolabeled heparin was completely inhibited by a 100-fold excess ofunlabeled heparin (data not shown).

The results, shown in Table II (as the mean values of duplicatewells±the range between the two values), indicated that the CNBrfragments containing the amino acids 71-100 (C3) and 1-56 and 112-170(C1,5) bound heparin to a similar extent. The CNBr fragment 171-196 alsobound more heparin than the control protein (thaumatin, a protein ofsimilar molecular weight and charge to rBPI₂₃).

The Asp-N fragments also demonstrated multiple heparin binding regionsin rBPI₂₃. As seen in Table II, the 57-104 Asp-N fragment bound thehighest amount of heparin, followed by the 1-38 and 116-193 fragments.These data, in combination with the CNBr fragment data, indicate thatthere are at least three separate heparin binding regions within rBPI₂₃,as demonstrated by chemically or enzymatically-generated fragments ofrBPI₂₃, with the highest heparin binding capacity residing withinresidues 71-100.

                  TABLE II                                                        ______________________________________                                        Heparin Binding of rBPI.sub.23 Fragments                                      Fragments   Region     cpm .sup.3 H-Heparin bound                             ______________________________________                                        CNBr Digest                                                                   C1, C5      1-56, 112-170                                                                            82,918 ± 4,462                                      C2          57-70      6,262 ± 182                                         C3           71-100    81,655 ± 3,163                                      C4          101-111    4,686 ± 4                                           C6          171-196    26,204 ± 844                                        Asp--N Digest                                                                 A1           1-38      17,002 ± 479                                        A2          15-38      3,042 ± 162                                         A3          39-56      8,664 ± 128                                         A4           57-104    33,159 ± 1,095                                      A6a         116-193    13,419 ± 309                                        rBPI.sub.23  1-193     51,222 ± 1,808                                      Thaumatin              7,432 ± 83                                          Wash Buffer            6,366 ± 46                                          ______________________________________                                    

EXAMPLE 4 Effect of BPI Proteolytic Fragments on an LAL Assay

BPI proteolytic fragments produced according to Example 1 were subjectedto a Limulus Amoebocyte Lysate (LAL) inhibition assay to determine LPSbinding properties of these fragments. Specifically, each of thefragments were mixed in Eppendorf tubes with a fixed concentration of E.coli 0113 LPS (4 ng/mL final concentration) and incubated at 37° C. for3 hours with occasional shaking. Addition controls comprising rBPI₂₃ at0.05 μg/mL were also tested. Following incubation, 360 μL of Dulbecco'sphosphate buffered saline (D-PBS; Grand Island Biological Co. (GIBCO),Long Island, N.Y.) were added per tube to obtain an LPS concentration of200 pg/mL for the LAL assay. Each sample was then transferred intoImmulon II strips (Dynatech, Chantilly, Va.) in volumes of 50 μl perwell.

Limulus amoebocyte Lysate (Quantitative Chromogenic LAL kit, WhitakerBioproducts, Inc., Walkersville, Md.) was added at 50 μL per well andthe wells were incubated at room temperature for 25 minutes. Chromogenicsubstrate was then added at a volume of 100 μL per well and was wellmixed. After incubation for 20 to 30 minutes at room temperature, thereaction was stopped with addition of 100 μL of 25% (v/v) acetic acid.Optical density at 405 nm was then measured in a multiplate reader(Model Vmax, Molecular Dynamics, Menlo Park, Calif.) with the resultsshown in FIG. 2 in terms of percent inhibition of LPS. In this Figure,the filled circle represents rBPI₂₃ ; the open circle represents Asp-Nfragment A3; the x represents Asp-N fragment A2; the filled squarerepresents Asp-N fragment A4; the filled triangle represents Asp-Nfragment A1A2; the open square represents Asp-N fragment A6a; the smallopen triangle represents CNBr fragment C3; and the small filled squarerepresents CNBr fragment C1/C5.

The CNBr digest fraction containing amino acid fragments 1-56 and112-170 inhibited the LPS-induced LAL reaction with an IC₅₀ ofapproximately 100 nM. This IC₅₀ is approximately 10-fold higher than theIC₅₀ for intact rBPI₂₃ (9 nM) in the same assay. The other CNBr digestfragments were found to be non-inhibitory.

A slightly different result was observed with fragments generated fromthe Asp-N digest, where three fragments were found to be inhibitory inthe LAL assay. The fragment corresponding to amino acids 116-193exhibited LAL inhibitory activity similar to intact rBPI₂₃ with completeinhibition of the LPS-induced LAL reaction at 15 nM. The fragmentscorresponding to amino acids 57-104 and 1-38 also inhibited the LALassay, but required 10-fold higher amounts. These results, incombination with the CNBr digest results, further supported theconclusion from previously-described experimental results that at leastthree regions of the rBPI₂₃ molecule have the ability to neutralize LBSactivation of the LAL reaction, with the most potent region appearing toexist within the 116-193 amino acid fragment.

Immunoreactivity studies of the proteolytic fragments of rBPI₂₃described in Example 1 were performed using ELISA assays. In suchassays, a rabbit polyclonal anti-rBPI₂₃ antibody, capable of blockingrBPI₂₃ bactericidal and LAL inhibition properties, and two different,non-blocking mouse anti-rBPI₂₃ monoclonal antibodies were used to probethe rBPI₂₃ proteolytic fragments. The polyclonal antibody was found tobe immunoreactive with the 116-193 and 57-104 Asp-N fragments and withthe 1-56 and 112-170 CNBr fragments, while the murine monoclonalantibodies reacted only with an Asp-N fragment representing residues1-14 of rBPI₂₃.

EXAMPLE 5 Preparation of 15-mer Peptides of BPI

In order to further assess the domains of biological activity detectedin the BPI fragment assays described in Examples 1-4, 15-mer syntheticpeptides comprised of 15 amino acids derived from the amino acidsequence of the 23 kD amino terminal fragment of BPI were prepared andevaluated for heparin-binding activity, activity in a Limulus AmoebocyteLysate Inhibition (LAL) assay and bactericidal activity. Specifically, aseries of 47 synthetic peptides were prepared, in duplicate, eachcomprising 15 amino acids and synthesized so that each peptide sharedoverlapping amino acid sequence with the adjacent peptides of the seriesby 11 amino acids, based on the sequence of rBPI₂₃ as previouslydescribed in copending U.S. patent application Ser. No. 08/093,202,filed Jul. 15, 1993.

Peptides were simultaneously synthesized according to the methods ofMaeji et al. (1990, Immunol. Methods 134: 23-33) and Gammon et al.(1991, J. Exp. Med. 173: 609-617), utlizing the solid-phase technologyof Cambridge Research Biochemicals Ltd. under license of CoselcoMimotopes Pty. Ltd. Briefly, the sequence of rBPI₂₃ (1-199) was dividedinto 47 different 15-mer peptides that progressed along the linearsequence of rBPI₂₃ by initiating a subsequent peptide every fifth aminoacid. This peptide synthesis technology allows for the simultaneoussmall scale synthesis of multiple peptides on separate pins in a 96-wellplate format. Thus, 94 individual pins were utilized for this synthesisand the remaining two pins (B,B) were subjected to the same steps as theother pins without the addition of activated FMOC-amino acids. Finalcleavage of the 15-mer peptides from the solid-phase pin supportemployed an aqueous basic buffer (sodium carbonate, pH 8.3). The uniquelinkage to the pin undergoes a quantitative diketopiperazine cyclizationunder these conditions resulting in a cleaved peptide with acyclo(lysylprolyl) moiety on the carboxyl-terminus of each peptide. Theamino-termini were not acetylated so that the free amino group couldpotentially contribute to anion binding reactions. An average of about15 μg of each 15-mer peptide was recovered per well.

EXAMPLE 6 Heparin Binding by 15mer Peptides of BPI

The BPI 15-mer peptides described in Example 5 were subjected to aheparin binding assay according to the methods described in Example 3.

The results of these experiments are shown in FIG. 3, expressed as thetotal number of cpm bound minus the cpm bound by control wells whichreceived blocking buffer only. These results indicated the existence ofthree distinct subsets of heparin-binding peptides representing separateheparin-binding functional domains in the rBPI₂₃ sequence. In the BPIsequence, the first domain was found to extend from about amino acid 21to about amino acid 55; the second domain was found to extend from aboutamino acid 65 to about amino acid 107; and the third domain was found toextend from about amino acid 137 to about amino acid 171. Material fromthe blank control pins showed no heparin binding effects.

EXAMPLE 7 Effect of 15-mer Peptides of BPI on an Limulus AmoebocyteLysate (LAL) Assay

The 15-mer peptides described in Example 5 were assayed for LPS bindingactivity using the LAL assay described in Example 4.

The results of these experiments are shown in FIG. 4. The data in FIG. 4indicated at least three major subsets of peptides representing threedistinct domains of the rBPI₂₃ protein having LPS-binding activityresulting in significant LAL inhibition. The first domain was found toextend from about amino acid 17 to about amino acid 55; the seconddomain was found to extend from about amino acid 73 to about amino acid99; and the third domain was found to extend from about amino acid 137to about amino acid 163. In addition, other individual peptides alsoexhibited LAL inhibition, as shown in the Figure. In contrast, materialfrom blank control pins did not exhibit any LPS neutralizing effects asmeasured by the LAL assay.

EXAMPLE 8 Bactericidal Effects of 15-mer Peptides of BPI

The 15-mer peptides described in Example 5 were tested for bactericidaleffects against the rough mutant strain of E. coli bacteria (J5) in aradial diffusion assay as described in Example 2. Products from theblank pins (B, B) were tested as negative controls.

The results of the assay are shown in FIG. 5. The only 15-mer peptidefound to have bactericidal activity was a peptide corresponding to aminoacids 85-99 of the BPI protein. As is seen in FIG. 5, the positivecontrol wells having varying amounts of rBPI₂₃ also showed bactericidalactivity, while the buffer and blank pin controls did not.

The results of these bactericidal assays, along with the heparin bindingand LAL assays described in the above Examples, indicate that thereexist discrete functional domains in the BPI protein.

The results shown in Examples 1-8 above indicate that rBPI₂₃ contains atleast three functional domains that contribute to the total biologicalactivity of the molecule. The first domain appears in the sequence ofamino acids between about 17 and 45 and is destroyed by Asp-N cleavageat residue 38. This domain is moderately active in both the inhibitionof LPS-induced LAL activity and heparin binding assays. The secondfunctional domain appears in the region of amino acids between about 65and 99 and its inhibition of LPS-induced LAL activity is diminished byCNBr cleavage at residue 70. This domain also exhibits the highestheparin binding capacity and contains the bactericidal peptide, 85-99.The third functional domain, between about amino acids 142 and 169, isactive in the inhibition of LPS-induced LAL stimulation assay andexhibits the lowest heparin binding capacity of the three regions.

EXAMPLE 9 Preparation of BPI Individual Functional Domain Peptides

Based on the results of testing the series of overlapping peptidesdescribed in Examples 5 through 8, BPI functional domain peptides fromeach of the functionally-defined domains of the BPI protein wereprepared by solid phase peptide synthesis according to the methods ofMerrifield, 1963, J. Am. Chem. Soc. 85: 2149 and Merrifield et al.,1966, Anal. Chem. 38: 1905-1914 using an Applied Biosystems, Inc. Model432 peptide synthesizer. BPI functional domain peptides were preparedhaving the amino acid sequences of portions of amino acid residues 1-199of BPI as set out in Table III below and designated BPI.2 through BPI.5and BPI.8.

                  TABLE III                                                       ______________________________________                                        BPI Individual Functional Domain Peptides                                     Polypeptide      Amino Acid Amino Acid                                                                             MW                                       No.     Domain   Region     Residues (daltons)                                ______________________________________                                        BPI.2   II       85-99      15       1828.16                                  BPI.3   II       73-99      27       3072.77                                  BPI.4   I        25-46      22       2696.51                                  BPI.5   III      142-163    22       2621.52                                  BPI.8   II       90-99      10       1316.8                                   ______________________________________                                    

EXAMPLE 10 Heparin Binding Activity by BPI Individual Functional DomainPeptides

BPI individual functional domain peptides BPI.2, BPI.3, and BPI.8, alongwith rBPI₂₁ Δcys were assayed for heparin binding activity according tothe methods described in Example 3. The results are shown in FIG. 6 andindicate that BPI.3 and rBPI₂₁ Δcys had moderate heparin bindingactivity and BPI.2 and BPI.8 had little or no heparin binding activity.

EXAMPLE 11 Heparin Neutralization Activity of BPI Individual FunctionalDomain Peptides

BPI functional domain peptides BPI.2, BPI.3, BPI.4, BPI.5, BPI.6, andBPI.8, along with rBPI₂₃ as a positive control, were assayed for theireffect on thrombin inactivation by ATIII/heparin complexes according tothe method of Example 3 in copending and co-assigned U.S. patentapplication Ser. No. 08/093,202, filed Jul. 15, 1993, incorporated byreference. Specifically, a Chromostrate™ anti-thrombin assay kit(Organon Teknika Corp., Durham, N.C.) was used to examine the inhibitionof purified thrombin by preformed ATIII/heparin complexes in plasma.

Briefly, the assay was performed in 96 well microtiter plates intriplicate with a final volume per well of 200 μL. Varyingconcentrations of the BPI functional domain peptides ranging from 1.0μg/mL to 100 μg/mL were assayed to determine their effect on thrombininhibition in the presence of pre-formed AThI/heparin complexes. Theorder of addition of assay components was as follows: 1) a dilutionseries of rBPI₂₃ or BPI functional domain peptides or thaumatin as acontrol protein, with final concentrations of 100, 50, 25, 10 and 1μg/well, diluted in PBS in a final volume of 50 μL; 2) 50 μL plasmadiluted 1:100 in a buffer supplied by the manufacturer; 3) 50 μlthrombin at 1 nKat/mL in a buffer supplied by the manufacturer; and 4)50 μL chromogenic substrate at a concentration of 1 μmol/mL in water.The reaction was allowed to proceed for 10 minutes at 37° C. and stoppedwith the addition of 50 μL 0.1M citric acid. The colorimetric reactionwas quantitated on a microplate reader as described in Example 3.

The results of these assays are shown in FIGS. 7a and 7b, which depictthe sample concentrations as weight or molar concentrationsrespectively. BPI functional domain peptides BPI.3 and BPI.5 each hadthe most significant heparin neutralization effects. In these assays,the control protein, thaumatin, showed no neutralizing effect and wasessentially equivalent to the buffer control at all proteinconcentrations.

EXAMPLE 12 LPS Neutralization Activity by LAL Assay of BPI IndividualFunctional Domain Peptides

BPI functional domain peptides BPI.2, BPI.3, and BPI.8, along withrBPI₂₃ as a positive control, were evaluated in the LAL assay accordingto the method of Example 4 herein to determine LPS binding andinhibition properties of these peptides. The experiments were performedessentially as described in Example 3 and the results are shown in FIGS.8a and 8b, which depict the sample concentrations as weight or molarconcentrations respectively. The results showed that BPI.3 had moderateLPS inhibition activity and that BPI.2 and BPI.8 had no significant LPSinhibition activity.

EXAMPLE 13 Bactericidal Activity Assay of BPI Individual FunctionalDomain Peptides

BPI functional domain peptides BPI.2, BPI.3, and BPI.8, along withrBPI₂₃ as a positive control, were tested for bactericidal effectsagainst E. coli J5 (rough) and E. coli 0111:B4 (smooth) bacteria in aradial diffusion assay according to the methods of Example 2. Theresults of these assays are depicted in FIGS. 9a-9d. These resultsdemonstrated that each of the BPI functional domain peptides BPI.2 andBPI.3 exhibited bactericidal activity while BPI.8 had little to nobactericidal activity. Each of the bactericidal peptides showingbactericidal activity tended to be more effective against the rough thanthe smooth E. coli strain.

In additional experiments, broth antibacterial assays were conducted tofurther determine the bactericidal activity of certain of the BPIpeptides. Specifically, either E. coli J5 (rough) or E. coli 0111:B4(smooth) bacteria were selected from single colonies on agar plates andused to inoculate 5 mL of Mueller Hinton broth and incubated overnightat 37° C. with shaking. The overnight culture was diluted (˜1:50) into 5mL fresh broth and incubated at 37° C. to log phase (˜3 hours). Bacteriawere pelleted for 5 minutes at 3000 rpm (1500 ×g). Bacterial pelletswere resuspended in 5 mL PBS and diluted to 2×10⁶ cells/mL in theMueller Hinton broth (wherein 1 OD₅₇₀ unit equals 1.25×10⁹ CFU/mL). TheBPI functional domain peptides to be tested were diluted to 200 μg/mL inbroth and serially diluted 2-fold in 96 well culture plates (100 μLvolume). All items were at 2-fold final concentration and experimentswere conducted in triplicate. Bacteria were added at 100 μL/well and theplates were incubated on a shaker at 37° C. for a 20 hour period. Theplates were then read on an ELISA plate multiple reader at 590 nm. Oneof the triplicate wells from each peptide concentration was selected forcolony forming unit (CFU) determination. A 30 μL aliquot was added to270 μL of PBS and further ten-fold serial dilutions were performed. Thena 50 μL aliquot was plated on tryptic soy agar and incubated overnight.Colonies were counted and final bacterial concentrations determined. Theresults of these assays are depicted in FIGS. 9e (for E. coli J5) and 9f(for E. coli 0111:B4). As shown in these Figures, BPI functional domainpeptide BPI.3 had significant anti-bacterial activity against E. coli J5bacteria and less activity against E. coli 0111:B4 bacteria.

EXAMPLE 14 Preparation of BPI Combination Functional Domain Peptides

Combination peptides were prepared using solid-phase chemistry asdescribed in Example 9. The sequences of these peptides are shown inTable IV. It will be noted that the peptides designated BPI.7, BPI.9 andBPI.10 represent partial or even multiple repeats of certain BPIsequences. Specifically, BPI.7 comprises a 20-mer consisting of aminoacid residues 90-99 repeated twice in a single linear peptide chain.BPI.10 comprises an approximately 50:50 admixture of a 25-mer(designated BPI.10.1; SEQ ID NO:55) and a 26-mer (designated BPI.10.2;SEQ ID NO:65) consisting of amino acid residues 94-99, 90-99, 90-99 and93-99, 90-99, 90-99, respectively, in a single linear peptide chain.BPI.9 comprises a 16-mer comprising amino acid residues 94-99 followedby residues 90-99 in a single linear peptide chain.

These peptides were used in each of the BPI activity assays described inExamples 10-13 above. In the heparin binding assay described in Example10 and shown in FIG. 6, BPI.7 had extremely high heparin bindingcapacity. In the heparin neutralization assay described in Example 11and shown in FIGS. 7a and 7b, BPI.7 had significant heparinneutralization effects compared with rBPI₂₃. In the LAL assay describedin Example 12 and shown in FIGS. 8a and 8b, BPI.7 had significant LPSinhibition properties. In bactericidal assays using radial diffusionplates as described in Example 13 and shown in FIGS. 9a-9d, each of theBPI functional domain peptides BPI.7, BPI.9 and BPI.10.1 and BPI.10.2exhibited bactericidal activity, and significant bactericidal activitywas also found for BPI.7, BPI.9 and BPI.10.1 and BPI.10.2 against bothrough and smooth variant strains of E. coli in broth assays. The BPI.10peptides exhibited the highest bactericidal activity observed againsteither bacterial strain.

These bactericidal activity results obtained with peptides BPI.7 andBPI.10 showed that a linear dimer (BPI.7) and a mixture of linearmultimers (BPI.10.1 and BPI.10.2) of the BPI domain II peptideKWKAQKRFLK (i.e., BPI.8, SEQ ID NO:8) had bactericidal activity againstE. coli strain J5, and that the monomer (BPI.8) showed essentially nobactericidal activity. Moreover, both the dimer and the multimerpeptides had higher bactericidal activity that of BPI.9, comprisingamino acids 94-99, 90-99. On the basis of these results, the additionalpeptides shown in Table IV were synthesized using the methods describedin Example 9.

                  TABLE IV                                                        ______________________________________                                        BPI Combination Functional Domain Peptides                                    BPI peptide                                                                           Amino Acid      Amino Acid                                                                              MW                                          No.     Region          Residues  (daltons)                                   ______________________________________                                        BPI.7   90-99, 90-99    20        2644.66                                     BPI.8   90-99           10        1316.8                                      BPI.9   94-99, 90-99    16        2131.34                                     BPI.10.1                                                                              94-99, 90-99,   25        3319.19                                             90-99                                                                 BPI.10.2                                                                              93-99, 90-99,   26        3447.32                                             90-99                                                                 BPI.13  148-161         14        1710.05                                     BPI.29  148-161, 148-161                                                                              28        3403.1                                      BPI.30  90-99, 148-161  24        3023.86                                     BPI.63  85-99, 148-161  29        3524.4                                      ______________________________________                                    

EXAMPLE 15 Bactericidal Activity of Combination Functional DomainPeptides

The BPI combination functional domain peptides described in Example 14were used in radial diffusion bactericidal assays essentially asdescribed in Examples 2 and 13 above. These results are shown in FIGS.10a-10e. The results shown in FIG. 10a demonstrate that BPI.8,comprising one copy of a domain II peptide (amino acids 90-99), had nodetectable bactericidal activity against E. coli J5 cells atconcentrations of 1000 μg/mL. In contrast, BPI.13, comprising one copyof a domain III monomer (amino acids 148-161) showed appreciablebactericidal activity at concentrations greater than 30 μg/mL. BPI.29,comprising two copies of a domain III monomer BPI.13, had greaterbactericidal activity, and BPI.30, comprising a linear combination ofthe domain II peptide BPI.8 and the domain III peptide BPI.13, showedthe highest bactericidal activity against J5 cells, approximating thatof BPI.

FIG. 10b shows the results of experiments with domain II peptidescomprising BPI.8, BPI.7 and BPI.10. (See also summary Table VIII.)Although BPI.8 showed no bactericidal activity against E. coli J5 cellsat concentrations of 1000 μg/mL, the combination peptides BPI.7 andBPI.10 showed high levels of bactericidal activity.

Additional experiments were performed using various other bacteria astarget cells to examine the range of bactericidal killing of these BPIfunctional domain peptides. FIG. 10c shows the results of radialdiffusion experiments using E. coli strain O7-K1. In these experiments,rBPI₂₃ showed no bactericidal activity at concentrations of 100 μg/mL,and low bactericidal activity even at concentrations of 1000 μg/mL.Similarly low levels of bactericidal activity were found with thepeptides BPI.8 comprising the domain II (DII) monomer and BPI.13comprising the domain III (DIII) monomer, although the amount ofactivity of BPI.13 was found to be higher than that of rBPI₂₃.Surprisingly, the domain II dimer BPI.7 and the domain II-domain III(DII-DIII) heterodimer BPI.30 showed high levels of bactericidalactivity, and the domain III dimer BPI.29 showed moderate bactericidalactivity. These results demonstrated that peptides of the functional BPIfunctional domain identified herein possess bactericidal activityqualitatively different from the bactericidal activity of the BPImolecule itself.

FIGS. 10d and 10e show results that further demonstrate that the homo-and heterodimers described herein have qualitatively and quantitativelydifferent bactericidal activity spectra of susceptible bacteria. FIG.10d shows the results of radial diffusion assays using Klebsiellapneumoniae bacteria. The DII-DIII heterodimer BPI.30 showed the highestamount of bactericidal activity against this bacteria, the DIIIhomodimer BPI.29 showed moderate levels of activity, and the DII dimer(BPI.7) and DIII monomer (BPI.13) showed low levels of activity. BPI.8,comprising the DII monomer, showed no bactericidal activity atconcentrations of 800 μg/mL, consistent with the lack of bactericidalactivity of this peptide seen with the E. coli strains tested.

FIG. 10e shows the levels of bactericidal activity found in radialdiffusion experiments using the Gram-positive bacterium Staphylococcusaureus. The DII-DIII heterodimer BPI.30 showed the highest amount ofbactericidal activity against this bacteria, the DIII homodimer BPI.29showed moderate levels of activity, and DII dimer (BPI.7) and the DIIImonomer (BPI.13) showed low levels of activity. BPI.8, comprising theDII monomer, showed no bactericidal activity at concentrations of 800μg/mL, consistent with the lack of bactericidal activity of this peptideseen with the other bacteria.

These results showed that the homo- and heterodimers disclosed hereinpossessed varying amounts of bactericidal activity, which varied bothwith regard to the amount of such activity and the minimum effectiveconcentration of the peptide necessary for bactericidal activity to bedetected. These results also showed that these peptides possessedquantitatively and, more surprisingly, qualitatively differentbactericidal activity than the BPI itself.

EXAMPLE 16 Additional Bactericidal Activity of BPI CombinationFunctional Domain Peptides

In light of the results of the experiments disclosed in Example 15, thebactericidal activity of domain II-domain III combination peptides werecompared with the bactericidal activity of each of the component BPIdomain II and domain III peptides, against a number of differentbacteria and other microorganisms. The following BPI functional domainpeptides as described above were used in radial diffusion bactericidalassays (Example 2) and broth bactericidal assays (Example 13)essentially as described in Example 15 above. These results are shown inFIGS. 11a-11q. These Figures show results of bactericidal assays usingthe following bacterial strains:

    ______________________________________                                                   BPI peptides tested                                                ______________________________________                                        Gram-negative bacteria                                                        Pseudomonas aeruginosa                                                                     BPI.8, BPI.13, BPI.30                                            E. coli O18:K1:H7                                                                          BPI.8, BPI.13, BPI.30                                            Klebsiella pneumoniae                                                                      BPI.8, BPI.13, BPI.30                                            E. coli O75  BPI.8, BPI.13, BPI.30                                            Serratia marcescens                                                                        BPI.8, BPI.13, BPI.30                                            Proteus mirabilis                                                                          BPI.2, BPI.13, BPI.30                                            Salmonella typhurium                                                                       BPI.23, BPI.30                                                   E. coli O86a:K61                                                                           BPI.23, BPI.30                                                   E. coli O4:K12                                                                             BPI.30                                                           Gram-positive bacteria                                                        Streptococcus pneumonia                                                                    BPI.29, BPI.30., BPI.48, BPI.55, BPI.13,                                      BPI.69                                                           Bacillus megaterium                                                                        BPI.2, BPI.7, BPI.45, BPI.46, BPI.47, BPI.48                     Staphylococcus aureus                                                                      BPI.7, BPI.8, BPI.10, BPI.13, BPI.30                             Fungi                                                                         Candida albicans                                                                           BPI.30, BPI.13, BPI.29, BPI.48, BPI.2                            ______________________________________                                    

The results of these experiments are summarized as follows. None of theBPI peptides tested showed any bactericidal activity against S.marcescens (FIG. 11f) or P. mirabilis (FIG. 11g). BPI.8 showed nobactericidal activity against any organism tested at concentrations upto about 2000 pmol. BPI.13 and BPI.30 showed bactericidal activityagainst P. aeruginosa (FIG. 11a), E. coli O18:K1:H7 (FIG. 11b), K.pneumoniae (FIG. 11c), and E. coli O 75 (FIG. 11d). Additionally, BPI.30showed bactericidal activity against S. typhurium (FIG. 11h), and, inbroth assays, E. coli O86a:K51 (FIG. 11j) and E. coli O4:K12 (FIG. 11k).BPI.23 showed bactericidal activity in a radial diffusion assay againstE. coli O86a:K61 (FIG. 11i). Additionally, BPI.30 showed bactericidalactivity against E. coli O86a:K61 in human serum (FIG. 11l).

The bactericidal capacity of BPI peptides provided by the invention wasalso tested against Gram-positive bacteria. Surprisingly, every BPIpeptide tested showed some bactericidal activity in radial diffusionassays using S. aureus (FIG. 11e), S. pneumoniae (FIG. 11m) and B.megaterium (FIG. 11n) at amounts ranging between about 20 and about 2000pmol. These results compared favorably with bactericidal activity of theantibiotics gentamicin and vancomycin (FIG. 11o).

Most surprisingly, one peptide, BPI.13, was found to have fungicidalactivity in a broth assay using C. albicans (FIGS. 11p and 11q). Asshown in these Figures, the activity of BPI.13 is clearlydistinguishable from the much lower activity levels of BPI.2, BPI.29,BPI.30, and BPI.48. These results demonstrate that the BPI functionaldomain peptides of the invention have antimicrobial activityqualitatively distinct from the activity previously reported for nativeBPI.

EXAMPLE 17 Heparin Neutralization Activity of BPI Combination FunctionalDomain Peptides

The in vitro and in vivo heparin neutralization capacity of the BPIcombination functional domain peptides prepared in Example 14 wasdetermined by assaying the ability of these peptides to counteract theinhibitory effect of heparin on clotting time of heparinized blood andplasma.

In vitro, the effect of BPI combination functional domain peptides wasdetermined on heparin-mediated lengthening of activated partial thrombintime (APTT). The APTT is lengthened by the presence of endogenous orexogenous inhibitors of thrombin formation, such as therapeuticallyadministered heparin. Thus, agents which neutralize the anti-coagulanteffects of heparin will reduce the AM measured by the test. Citratedhuman plasma (200 μL) was incubated for 1 minute at 37° C. with either15 μL of diluent (0.15M NaCl, 0.1M Tris-HCl, pH 7.4) or 15 μL of thediluent also containing 25 μg/mL heparin (187 units/mg). Variousconcentrations (from 0.0 to 56 μg/mL) of rBPI₂₃, rBPI₂₁ Δcys, or BPIcombination peptides BPI.29 (the DIII homodimer) and BPI.30 (heterodimerDII+DIII) in a volume of 15 μL were added, followed immediately by 100μL of thrombin reagent (Catalog No.: 845-4, Sigma Chemical Co., St.Louis, Mo.). Clotting time (thrombin time) was measured using a BBLFibrometer (Becton Dickenson Microbiology Systems, Cockeysville, Md.).The results are shown in FIGS. 12a, 12b and 12e. FIG. 12a shows therelative decrease caused by addition of varying amounts of rBPI₂₃ orrBPI₂₁ Δcys to the heparin-prolonged APTT. These results establish thateach of these BPI-related proteins inhibits the heparin-mediatedlengthening of APTT. FIG. 12b shows that the BPI combination peptidesBPI.29 and BPI.30 also inhibit the heparin-mediated lengthening of APTT.FIG. 12e illustrates the results obtained with BPI.30 on a non-logscale. FIG. 12g shows that BPI.29, BPI.30, and BPI.7 have the greatesteffect on the clotting time of heparinized blood in the assay. BPI.3 andrBPI₂₃ show a smaller effect, and BPI.14, BPI.2, BPI.4, BPI.5, BPI.7,and rLBP₂₅, rBPI and rBPI₂₁ Δcys all show less of a decrease in clottingtimes of heparinized blood in this assay.

The in vivo effect of exemplary BPI combination peptides on APTT inheparinized rats was determined and compared with the in vivo effect ofrBPI₂₃. APTT is lengthened by the presence of endogenous or exogenousinhibitors of thrombin formation, such as therapeutically administeredheparin. Agents which neutralize the anticoagulant effects of heparinwill reduce the APTT as measured by this test. Sprague-Dawley ratshoused under NIH guidelines were administered with 100 U/kg heparin bybolus intravenous injections via the animals' tail vein followed 5minutes later by administration of varying amounts of test or controlprotein as compared with rBPI₂₃. The APTT was then determined from bloodsamples collected from the abdominal aorta 2 minutes after theadministration of the test or control protein. The APTT of untreatedanimals, as well as animals treated only with a BPI peptide, was alsodetermined. FIG. 12c shows the dose dependence of rBPI₂₃ inhibition ofheparin-mediated lengthening of partial thromboplastin time, and thatadministration of about 5 mg/kg results in a APTT of the heparinized andBPI-treated animals that is almost the same as the untreated controlanimals. The results of similar experiments shown in FIG. 12ddemonstrate that the unrelated protein thaumatin has no effect on APTTtimes in heparinized animals. The administration of BPI.10 peptideresults in a APTT in heparinized animals that is essentially the same asthe APTT in control animals treated with BPI.10 alone. Similar resultsusing BPI.30 were also obtained (FIG. 12f).

These results show that BPI functional domain combination peptides(e.g., BPI.10 and BPI.30) and rBPI₂₃ effectively neutralize heparininhibition of coagulation proteases. Based on these characteristics, BPIcombination functional domain peptides of the invention are projected tobe useful in the clinical neutralization of heparin anticoagulanteffects in dosages generally corresponding functionally to thoserecommended for protamine sulfate, but are not expected to possess thesevere hypotensive and anaphylactoid effects of that material.

EXAMPLE 18 Preparation and Functional Activity Analysis of BPISubstitution Variant Functional Domain Peptides

The results obtained above with peptides from functional domains II andIII prompted a further effort to determine the functionally-importantamino acid residues within these peptides. Accordingly, a series ofpeptides comprising the amino acid sequences of domains II and III wereprepared in which one of the amino acids in the sequence was substitutedwith an alanine residue. Diagrams of the domain peptides used in thesubstitution experiments are shown in FIG. 13 (domain II;IKISGKWKAQKRFLK, SEQ ID No.:7) and FIG. 14 (domain III; KSKVGWLIQLFHKK,SEQ ID No.:13). These peptide series were then tested for heparinbinding affinity (K_(d)), heparin binding capacity (Hep-CAP), LPSneutralization as determined using the Limulus Ameboctye Lysate assay(LAL), and bactericidal activity against E. coli J5 using the radialdiffusion assay (RAD), each assay as performed as described in theExamples above.

The results, shown in Table V (domain II) and Table VI (domain III), areexpressed in terms of the fold difference in activity in each of theseassays (except for the LAL assay where relative differences are noted)between the BPI functional domain II and domain III peptides and eachalanine substituted variant peptide thereof.

For domain II peptides, most alanine-substituted peptides showed anapproximately 2- to 10-fold reduction in bactericidal activity in theradial diffusion assay. Exceptions to this overall pattern includeBPI.19 (Gly₈₉ →Ala₈₉), BPI.22 (Lys₉₂ →Ala₉₂), BPI.23 (Gln₉₄ →Ala₉₄) andBPI.24 (Lys₉₅ →Ala₉₅). In contrast, most alanine-substituted peptidesshowed no difference in the LAL assay; BPI.17 (Ile₈₇ →Ala₈₇) and BPI.21(Trp₉₁ →Ala₉₁) showed a moderate and large decrease in activity,respectively, in this assay. For BPI.21, these results were consistentwith the more than 10-fold reduction in bactericidal activity found forthis peptide, indicating that amino acid 91 (a tryptophan residue in thenative sequence) may be particularly important in conferring biologicalactivity on the peptide.

The effect of alanine substitution on heparin binding and capacity was,in almost all cases, no more than 2-fold more or less than theunsubstituted peptide. One exception was the heparin binding capacity ofBPI.21, which was 4-fold lower than the unsubstituted peptide. Thisfurther supports the earlier results on the particular sensitivity ofthe various activities of these peptides to substitution at Trp₉₁. Inmost cases, the effect on both the K_(d) of heparin binding and heparinbinding capacity was consistent and of about the same magnitude. In someinstances, the heparin binding capacity of the substituted peptidedecreased, although the K_(d) increased slightly (BPI.18; Ser₈₈ →Ala₈₈),or decreased slightly (BPI.24). There were also instances where capacitywas unchanged even though the K_(d) increased (BPI.20; Lys₉₀ →Ala₉₀) ordecreased (BPI.19). In one instance the affinity remained unaffected andthe capacity decreased almost 2-fold (BPI.25; Arg₉₆ →Ala₉₆).

These results indicated the existence of at least one critical residuein the domain II sequence (Trp₉₁), and that the activities of the domainII peptides were for the most part only minimally affected by alaninesubstitution of the other domain II amino acid residues.

For domain III peptides, most alanine-substituted peptides showed anapproximately 2- to 5-fold reduction in bactericidal activity in theradial diffusion assay. Exceptions to this overall pattern includeBPI.35 (Gly₁₅₂ →Ala₁₅₂), BPI.39 (Gln₁₅₆ →Ala₁₅₆), BPI.42 (His₁₅₉→Ala₁₅₉) and BPI.44 (Lys₁₆₁ →Ala₁₆₁). Most alanine-substituted peptidesshowed no difference in the LAL assay; BPI.31 (Lys₁₄₈ →Ala₁₄₈), BPI.32(Ser₁₄₉ →Ala₁₄₉), BPI.33 (Lys₁₅₀ →Ala₁₅₀), and BPI.34 (Val₁₅₁ →Ala₁₅₁)showed a moderate decrease in LPS-binding activity, and BPI.36 (Trp₁₅₃→Ala₁₅₃) and BPI.40 (Leu₁₅₇ →Ala₁₅₇) showed a large decrease inLPS-binding activity in this assay. For both BPI.36 and BPI.40, theseresults were consistent with the approximately 5-fold reduction inbactericidal activity found for these peptides, indicating that thehydrophobic amino acids Trp₁₅₃ and Leu₁₅₇ in the native sequence may beparticularly important in conferring biological activity on the peptide.

Effects of alanine substitution on heparin binding and capacity were ofsimilar magnitude, being no more than about 5-fold more or less than theunsubstituted peptide. In almost every case, the type of effect ofalanine substitutions on both the K_(d) of heparin binding and heparinbinding capacity was consistent and of about the same magnitude, unlikethe findings with the domain II alanine substitution peptides. In oneinstance (BPI.42; His₁₅₉ →Ala₁₅₉), the heprin binding capacity wasunaffected although the K_(d) declined slightly (1.2-fold). In only oneinstance was the K_(d) of heparin binding and heparin capacity increasedslightly (BPI.35; Gly₁₅₂ →Ala₁₅₃); an increase of only 10% was found.

Like the results found with the domain II alanine-substitution peptides,these results indicated the existence of at least one critical residuein the domain III sequence (Trp₁₅₃), and possibly at least one other(Leu₁₅₇). The results also showed that, unlike the domain IIalanine-substituted peptides, almost one-half of the substitutionsresulted in at least a 2-fold difference in the activities tested. In 6cases, all four of the tested activities decreased, and in 10 instancesbactericidal activity, the K_(d) of heparin binding and heparin capacitydecreased. In only one instance (BPI.35, Gly₁₅₂ →Ala₁₅₂) was theactivity in the bactericidal, heparin binding K_(d) and heparin capacityassays found to have increased, albeit slightly.

These results indicate that alanine replacement of the hydrophobic aminoacid residues Trp₉₁, Trp₁₅₃ and Leu₁₅₇ have the greatest effect on theactivities of these BPI functional domain substitution peptides. Thisresult is unexpected in light of the cationic nature of rBPI₂₃. In fact,domain II alanine substitution peptides in which lysine is replacedeither by alanine or phenylalanine showed dramatic increases in activity(e.g., BPI.24, BPI.73).

                                      TABLE V                                     __________________________________________________________________________    BPI DOMAIN II ALANINE SUBSTITUTION PEPTIDES                                                                     FOLD CHANGE IN ACTIVITY                                                       RAD LAL                                                                              HEPK.sub.d                                                                        HEPCAP                           __________________________________________________________________________    BPI.2                                                                             I K I S G K W K A Q K R F L K                                             BPI.15                                                                            A                             ↓2.2                                                                       =  ↓1.1                                                                   ↓1.4                          BPI.16                                                                              A                           ↓1.8                                                                       =  ↓1.5                                                                   ↓1.6                          BPI.17  A                         ↓4.5                                                                       ↓                                                                         ↓1.3                                                                   ↓1.8                          BPI.18    A                       ↓1.6                                                                       =  ↑1.1                                                                    ↓1.3                          BPI.19      A                     ↑1.4                                                                        =  ↓1.3                                                                   =1.0                                 BPI.20        A                   ↓1.1                                                                       =  ↑1.4                                                                    =1.0                                 BPI.21          A                 ↓10.4                                                                      ↓↓                                                                 ↓1.5                                                                   ↓4.0                          BPI.22            A               =1.0                                                                              =  ↓1.1                                                                   ↓1.5                          BPI.23                A           ↑2.2                                                                        =  ↑2.0                                                                    ↑1.4                           BPI.24                  A         ↑3.8                                                                        =  ↓2.1                                                                   ↑2.1                           BPI.25                    A       ↓3.8                                                                       =  =1.0 ↓1.9                     BPI.26                      A     ↓4.0                                                                       =  ↓1.5                                                                   ↓1.8                          BPI.27                        A   ↓2.5                                                                       =  ↓1.7                                                                   ↓1.7                          BPI.28                          A ↓2.4                                                                       =  ↓1.3                                                                   ↓1.3                          __________________________________________________________________________

                                      TABLE VI                                    __________________________________________________________________________    BPI DOMAIN III ALANINE SUBSTITUTION PEPTIDES                                                                  FOLD REDUCTION IN ACTIVITY                                                    RAD                                                                              LAL                                                                              HEPK.sub.d                                                                        HEPCAP                              __________________________________________________________________________    BPI.13                                                                            K S K V G W L I Q L F H K K                                               BPI.31                                                                            A                           ↓2.3                                                                      ↓                                                                         ↓3.9                                                                       ↓1.8                         BPI.32                                                                              A                         ↓1.5                                                                      ↓                                                                         ↓2.9                                                                       ↓1.7                         BPI.33  A                       ↓1.4                                                                      ↓                                                                         ↓2.0                                                                       ↓1.6                         BPI.34    A                     ↓2.2                                                                      ↓                                                                         ↑1.9                                                                        ↓1.8                         BPI.35      A                   ↑1.5                                                                       =  ↑1.1                                                                        ↑1.1                          BPI.36        A                 ↓4.9                                                                      ↓↓                                                                 ↓2.6                                                                       ↓4.6                         BPI.37          A               ↓3.8                                                                      =  ↓5.2                                                                       ↓2.7                         BPI.38            A             ↓5.0                                                                      =  ↓1.7                                                                       ↓1.9                         BPI.39                A         ↑1.1                                                                       =  ↓1.3                                                                       ↓1.1                         BPI.40                  A       ↑5.2                                                                       ↓↓                                                                 ↓1.8                                                                       ↓2.3                         BPI.41                    A     ↓4.3                                                                      =  ↓2.1                                                                       ↓3.1                         BPI.42                      A   ↑2.2                                                                       =  ↓1.2                                                                       =1.0                                BPI.43                        A ↓1.3                                                                      =  ↓2.0                                                                       ↓1.3                         BPI.44                          ↑1.2                                                                       =  ↓1.7                                                                       ↓1.1                         __________________________________________________________________________

EXAMPLE 19 Summary of Biological Activity of BPI Functional DomainPeptides

The distribution of the peptides into construct categories is presentedin Table VII below.

The BPI functional domain peptides of this invention, or representativesubsets thereof, have been assayed for the following biologicalactivities: bactericidal activity against Gram-negative andGram-positive bacteria, and against certain other microorganisms; LPSbinding and neutralization activities; and heparin binding and heparinneutralization activities.

BPI functional domain peptides were assayed for bactericidal activity onE. coli J5 bacteria and for heparin binding as described in Examples 8and 6, respectively. The assay results for exemplary peptides of thepresent invention are summarized in Table VIII for the Gram-negativebacteria E. coli J5 (rough) and E. coli O113 (smooth) and theGram-positive bacteria S. aureus. The bactericidal activities areexpressed as the amount of peptide (pmol/well and μg/well) required togenerate a 30 mm² bactericidal zone.

                  TABLE VII                                                       ______________________________________                                              Seq                                                                     BPI   ID                                                                      Peptide                                                                             No.    Peptide Sequence                                                 ______________________________________                                        I. BPI individual functional domain peptides                                  Domain                                                                        Peptides                                                                      BPI.1  4     QQGTAALQKELKRIK                                                  BPI.4  3     LQKELKRIKIPDYSDSFKIKHL                                           BPI.14                                                                               2     GTAALQKELKRIKIPDYSDSFKIKHLGKGH                                   BPI.54                                                                               5     GTAALQKELKRIKIP                                                  Domain                                                                        II                                                                            Peptides                                                                      BPI.2  7     IKISGKWKAQKRFLK                                                  BPI.3 11     NVGLKFSISNANIKISGKWKAQKRFLK                                      BPI.8  8     KWKAQKRFLK                                                       Domain                                                                        III                                                                           Peptides                                                                      BPI.5 67     VHVHISKSKVGWLIQLFHKKIE                                           BPI.11                                                                              13     KSKVWLIQLFHKK                                                    BPI.12                                                                              14     SVHVHISKSKVGWLIQLFHKKIESALRNK                                    BPI.13                                                                              15     KSKVGWLIQLFHKK                                                   BPI.55                                                                              61     GWLIQLFHKKIESALRNKMNS                                            II. Linear and branched-chain combination peptides                            Domain                                                                        II                                                                            Peptides                                                                      BPI.7 54     KWKAQKRFLKKWKAQKRFLK                                             BPI.9 51     KRFLKKWKAQKRFLK                                                  BPI.  55     KRFLKKWKAQKRFLKKWKAQKRFLK                                        10.1                                                                          BPI.  65     QKRFLKKWKAQKRFLKKWKAQKRFLK                                       10.2                                                                          MAP.1        β--Ala--Nα,Nε-- Nα,Nε(BPI.2)                 Lys!Lys                                                          Domain                                                                        III                                                                           Peptides                                                                      BPI.29                                                                              56     KSKVGWLIQLFHKKKSKVGWLIQLFHKK                                     MAP.2        β--Ala--Nα,Nε-- Nα,Nε(BPI.13                 )Lys!Lys                                                         III. Single amino acid substitution peptides                                  Domain                                                                        II                                                                            Peptides                                                                      BPI.15                                                                              16     AKISGKWKAQKRFLK                                                  BPI.16                                                                              17     IAISGKWKAQKRFLK                                                  BPI.17                                                                              18     IKASGKWKAQKRFLK                                                  BPI.18                                                                              19     IKIAGKWKAQKRFLK                                                  BPI.19                                                                              20     IKISAKWKAQKRFLK                                                  BPI.20                                                                              21     IKISGAWKAQKRFLK                                                  BPI.21                                                                              22     IKISGKAKAQKRFLK                                                  BPI.22                                                                              23     IKISGKWAAQKRFLK                                                  BPI.23                                                                              24     IKISGKWKAAKRFLK                                                  BPI.24                                                                              25     IKISGKWKAQARFLK                                                  BPI.25                                                                              26     IKISGKWKAQKAFLK                                                  BPI.26                                                                              27     IKISGKWKAQKRALK                                                  BPI.27                                                                              28     IKISGKWKAQKRFAK                                                  BPI.28                                                                              29     IKISGKWKAQKRFLA                                                  BPI.61                                                                              48     IKISGKFKAQKRFLK                                                  BPI.73                                                                              62     IKISGKWKAQFRFLK                                                  BPI.77                                                                              72     IKISGKWKAQWRFLK                                                  BPI.79                                                                              73     IKISGKWKAKKRFLK                                                  BPI.81                                                                              75     IKISGKWKAFKRFLK                                                  Domain                                                                        III                                                                           Peptides                                                                      BPI.31                                                                              33     ASKVGWLIQLFHKK                                                   BPI.32                                                                              34     KAKVGWLIQLFHKK                                                   BPI.33                                                                              35     KSAVGWLIQLFHKK                                                   BPI.34                                                                              36     KSKAGWLIQLFHKK                                                   BPI.35                                                                              37     KSKVAWLIQLFHKK                                                   BPI.36                                                                              38     KSKVGALIQLFHKK                                                   BPI.37                                                                              39     KSKVGWAIQLFHKK                                                   BPI.38                                                                              40     KSKVGWLAQLFHKK                                                   BPI.39                                                                              41     KSKVGWLIALFHKK                                                   BPI.40                                                                              42     KSKVGWLIQAFHKK                                                   BPI.41                                                                              43     KSKVGWLIQLAHKK                                                   BPI.42                                                                              44     KSKVGWLIQLFAKK                                                   BPI.43                                                                              45     KSKVGWLIQLFHAK                                                   BPI.44                                                                              46     KSKVGWLIQLFHKA                                                   BPI.82                                                                              76     KSKVGWLIQLWHKK                                                   BPI.85                                                                              79     KSKVLWLIQLFHKK                                                   BPI.86                                                                              80     KSKVGWLILLFHKK                                                   BPI.87                                                                              81     KSKVGWLIQLFLKK                                                   BPI.91                                                                              86     KSKVGWLIFLFHKK                                                   BPI.92                                                                              87     KSKVGWLIKLFHKK                                                   BPI.94                                                                              89     KSKVGWLIQLFFKK                                                   BPI.95                                                                              90     KSKVFWLIQLFHKK                                                   BPI.96                                                                              91     KSKVGWLIQLFHKF                                                   BPI.97                                                                              92     KSKVKWLIQLFHKK                                                   IV. Double amino acid substitution peptides                                   Domain                                                                        II                                                                            Peptides                                                                      BPI.45                                                                              31     IKISGKWKAAARFLK                                                  BPI.56                                                                              47     IKISGKWKAKQRFLK                                                  BPI.59                                                                              30     IKISGAWAAQKRFLK                                                  BPI.60                                                                              32     IAISGKWKAQKRFLA                                                  BPI.88                                                                              82     IKISGKWKAFFRFLK                                                  Domain                                                                        III                                                                           Peptides                                                                      BPI.100                                                                             94     KSKVKWLIKLFHKK                                                   Va. Double amino acid substitution/combination peptides                       Domain                                                                        II                                                                            Peptides                                                                      BPI.46                                                                              57     KWKAAARFLKKWKAQKRFLK                                             BPI.47                                                                              58     KWKAQKRFLKKWKAAARFLK                                             BPI.48                                                                              59     KWKAAARFLKKWKAAARFLK                                             Vb. Multiple amino acid substitution/combination peptides                     Domain                                                                        II                                                                            Peptides                                                                      BPI.69                                                                              60     KWKAAARFLKKWKAAARFLKKWKAAARFLK                                   BPI.99                                                                              93     KWKAQWRFLKKWKAQWRFLKKWKAQWRFLK                                   BPI.101                                                                             95     KSKVKWLIKLFFKFKSKVKWLIKLFFKF                                     VIa. Atypical amino acid substitution peptides                                Domain                                                                        II                                                                            Peptides                                                                      BPI.66                                                                              49     IKISGKW.sub.D KAQKRFLK                                           BPI.67                                                                              50     IKISGKA.sub.β-(1-naphthyl) KAQKRFLK                         BPI.70                                                                              63     IKISGKWKA.sub.β-(3-pyridyl) QKRFLK                          BPI.71                                                                              66     A.sub.D A.sub.D IKISGKWKAQKRFLK                                  BPI.72                                                                              64     IKISGKWKAQKRA.sub.β-(3-pyridyl)                             BPI.76                                                                              71     IKISGKWKAQF.sub.D RFLK                                           BPI.80                                                                              74     IKISGKWKAQA.sub.β-(1-naphthyl) RFLK                         Domain                                                                        III                                                                           Peptides                                                                      BPI.83                                                                              77     KSKVGA.sub.β-(1-naphthyl) LIQLFHKK                          VIb. Atypical amino acid double substitution peptides                         Domain                                                                        II                                                                            Peptides                                                                      BPI.84                                                                              78     IKISGKA.sub.β-(1-naphthyl) KAQFRFLK                         BPI.89                                                                              84     IKISGKA.sub.β-(1-naphthyl) KAFKRFLK                         VIc. Atypical amino acid triple substitution peptides                         Domain                                                                        II                                                                            Peptides                                                                      BPI.90                                                                              85     IKISGKA.sub.β-(1-naphthyl) KAFFRFLK                         VII. Cyclized peptides                                                        Domain                                                                        II                                                                            Peptides                                                                      BPI.58                                                                               9     CIKISGKWKAQKRFLK                                                 BPI.65                                                                              10     CIKISGKWKAQKRFLKC                                                oxidiz-                                                                       ed                                                                            BPI.65                                                                              10     CIKISGKWKAQKRFLKC                                                reduced                                                                       VIIIa. Interdomain combination peptides                                       Domain                                                                        II-                                                                           Domain                                                                        III                                                                           Peptides                                                                      BPI.30                                                                              52     KWKAQKRFLKKSKVGWLIQLFHKK                                         BPI.63                                                                              53     IKISGKWKAQKRFLKKSKVGWLIQLFHKK                                    BPI.74                                                                              70     KSKVGWLIQLFHKKKWKAQKRFLK                                         VIIIb. Interdomain combination multiple substitution peptides                 Domain                                                                        II-                                                                           Domain                                                                        III                                                                           Peptides                                                                      BPI.102                                                                             96     KWKAQFRFLKKSKVGWLILLFHKK                                         VIIIc. Atypical ainino acid double substitution/interdomain combination       peptides                                                                      Domain                                                                        II-                                                                           Domain                                                                        III                                                                           Peptides                                                                      BPI.93                                                                              88     IKISGKA.sub.β-(1-naphthyl) KAQFRFLKKSKVGWLIQLFHKK           BPI.98                                                                              83     IKISGKA.sub.β-(1-naphthyl) KAQFRFLKKSKVGWLIFLFHKK           ______________________________________                                    

                                      TABLE VIII                                  __________________________________________________________________________    Bactericidal Activity.sup.a                                                   BPI  E. coli J5 E. coli O111:B4                                                                          S. aureus                                          Peptide                                                                            (pmol/well)                                                                         (μg/well)                                                                       (pmol/well)                                                                         (μg/well)                                                                       (pmol/well)                                                                         (μg/well)                                 __________________________________________________________________________    BPI.1                                                                              .sup. --.sup.b                                                                      --   --    --   --  --                                             BPI.2                                                                              >2733.5                                                                             >5   --    --   --  --                                             BPI.3                                                                              696   2.14 --    --    N.T..sup.c                                                                       N.T.                                           BPI.4                                                                              --    --   --    --   --  --                                             BPI.5                                                                              398   1.05 >1904 >5   N.T.                                                                              N.T.                                           BPI.6                                                                              --    --   --    --   --  --                                             BPI.7                                                                              175   0.46 >1890.6                                                                             >5   >1890.6                                                                           >5                                             BPI.8                                                                              >3797.1                                                                             >5   --    --   N.T.                                                                              N.T.                                           BPI.9                                                                              479   1.02 >2345.9                                                                             >5   N.T.                                                                              N.T.                                           BPI.10                                                                             102   0.41 697   2.76 N.T.                                                                              N.T.                                           BPI.11                                                                             638   1.06 --    --   N.T.                                                                              N.T.                                           BPI.12                                                                             525   1.78 --    --   N.T.                                                                              N.T.                                           BPI.13                                                                             441   0.75 >2923.9                                                                             >5   >2923.9                                                                           >5                                             BPI.14                                                                             --    --   --    --   N.T.                                                                              N.T.                                           BPI.15                                                                             >2797.8                                                                             >5   --    --   N.T.                                                                              N.T.                                           BPI.16                                                                             >2821.5                                                                             >5   --    --   N.T.                                                                              N.T.                                           BPI.17                                                                             >2807.2                                                                             >5   --    --   N.T.                                                                              N.T.                                           BPI.18                                                                             >2757.6                                                                             >5   --    --   N.T.                                                                              N.T.                                           BPI.19                                                                             >2712.8                                                                             >5   --    --   N.T.                                                                              N.T.                                           BPI.20                                                                             >2821.5                                                                             >5   --    --   N.T.                                                                              N.T.                                           BPI.21                                                                             >2917 >5   --    --   N.T.                                                                              N.T.                                           BPI.22                                                                              >2821.50                                                                           >5   --    --   N.T.                                                                              N.T.                                           BPI.23                                                                             1330  2.36  >2821.15                                                                           >5   N.T.                                                                              N.T.                                           BPI.24                                                                             655   1.16  >2821.50                                                                           >5   N.T.                                                                              N.T.                                           BPI.25                                                                             >2866.8                                                                             >5   --    --   N.T.                                                                              N.T.                                           BPI.26                                                                             >2852.1                                                                             >5   --    --   N.T.                                                                              N.T.                                           BPI.27                                                                             >2797.8                                                                             >5   --    --   N.T.                                                                              N.T.                                           BPI.28                                                                             >2821.5                                                                             >5   --    --   N.T.                                                                              N.T.                                           BPI.29                                                                             442   1.5  >1469.2                                                                             >5   >1469.2                                                                           >5                                             BPI.30                                                                              76   0.23 608   1.84 1216                                                                              3.68                                           BPI.31                                                                             938   1.55 --    --   N.T.                                                                              N.T.                                           BPI.32                                                                             614   1.04 --    --   N.T.                                                                              N.T.                                           BPI.33                                                                             575   0.95 --    --   N.T.                                                                              N.T.                                           BPI.34                                                                             916   1.54 --    --   N.T.                                                                              N.T.                                           BPI.35                                                                             263   0.45 --    --   N.T.                                                                              N.T.                                           BPI.36                                                                             1652  2.64 --    --   N.T.                                                                              N.T.                                           BPI.37                                                                             1284  2.14 --    --   N.T.                                                                              N.T.                                           BPI.38                                                                             1698  2.83 --    --   N.T.                                                                              N.T.                                           BPI.39                                                                             316   0.52 --    --   N.T.                                                                              N.T.                                           BPI.40                                                                             1760  2.94 --    --   N.T.                                                                              N.T.                                           BPI.41                                                                             2465  4.03 --    --   N.T.                                                                              N.T.                                           BPI.42                                                                             265   0.44 >3041.3                                                                             >5   N.T.                                                                              N.T.                                           BPI.43                                                                             729   1.21 >3024.8                                                                             >5   N.T.                                                                              N.T.                                           BPI.44                                                                             481   0.8  2983  4.93 N.T.                                                                              N.T.                                           BPI.45                                                                             1302  2.23 >1696.7                                                                             >5   >1696.7                                                                           >5                                             BPI.46                                                                             186   0.47 >1811.2                                                                             >5   >1811.2                                                                           >5                                             BPI.47                                                                              98   0.25 577   1.46 >2461.9                                                                           >5                                             BPI.48                                                                              42   0.1  254   0.61 >1390.4                                                                           >5                                             BPI.49                                                                             N.T.  N.T. N.T.  N.T. N.T.                                                                              N.T.                                           BPI.50                                                                             N.T.  N.T. N.T.  N.T. N.T.                                                                              N.T.                                           BPI.51                                                                             N.T.  N.T. N.T.  N.T. N.T.                                                                              N.T.                                           BPI.52                                                                             N.T.  N.T. N.T.  N.T. N.T.                                                                              N.T.                                           BPI.53                                                                             --    --   --    --   N.T.                                                                              N.T.                                           BPI.54                                                                             --    --   --    --   N.T.                                                                              N.T.                                           BPI.55                                                                             299   0.75 >1592.2                                                                             >5   >1592.2                                                                           >5                                             BPI.56                                                                             1387  2.54 --    --   --  --                                             BPI.57                                                                             514   1.05 --    --   --  --                                             BPI.58                                                                             1050  2.03 --    --   --  --                                             BPI.59                                                                             >2312.3                                                                             >5   --    --   --  --                                             BPI.60                                                                             >2136.5                                                                             >5   --    --   --  --                                             BPI.61                                                                             >2093.5                                                                             >5   --    --   --  --                                             BPI.62                                                                             N.T.  N.T. N.T.  N.T. N.T.                                                                              N.T.                                           BPI.63                                                                              87   0.31 512   1.8  >1006.3                                                                           >5                                             BPI.64                                                                             N.T.  N.T. N.T.  N.T. N.T.                                                                              N.T.                                           BPI.65                                                                             895   1.82 --    --   >3118                                                                             >5                                             oxidized                                                                      BPI.65                                                                             1362  2.77 --    --   --  --                                             reduced                                                                       BPI.66                                                                             >3496.7                                                                             >5   --    --   --  --                                             BPI.67                                                                             >1901.8                                                                             >5   --    --   --                                                 BPI.68                                                                             N.T.  N.T. N.T.  N.T. N.T.                                                                              N.T.                                           BPI.69                                                                              57   0.21 244   0.88 1058                                                                              3.83                                           BPI.70                                                                             --    --   --    --   --  --                                             BPI.71                                                                             2297  4.53 --    --   --  --                                             BPI.72                                                                             >1911.2                                                                             >5   --    --   --  --                                             BPI.73                                                                              57   0.11 >1810.9                                                                             >5   >1810.9                                                                           >5                                             BPI.74                                                                             732   2.21 >2148.2                                                                             >5   >2148.2                                                                           >5                                             BPI.75                                                                               2030.8                                                                            4.96 --    --   >2030.8                                                                           >5                                             BPI.76                                                                             >3906.5                                                                             >5   --    --   --  --                                             BPI.77                                                                             455   0.85 --    --     1684.5                                                                          3.15                                           BPI.78                                                                             N.T.  N.T. N.T.  N.T. N.T.                                                                              N.T.                                           BPI.79                                                                             >2282.9                                                                             >5   --    --   --  --                                             BPI.80                                                                             655   1.24 --    --   >1975.4                                                                           >5                                             BPI.81                                                                             284   0.52 >2344.9                                                                             >5   >2344.9                                                                           >5                                             BPI.82                                                                             171   0.32 >1197.8                                                                             >5   >1197.8                                                                           >5                                             BPI.83                                                                             155   0.27 >2033.5                                                                             >5   >2033.5                                                                           >5                                             BPI.84                                                                              12   0.02 >2016.9                                                                             >5   >2016.9                                                                           >5                                             BPI.85                                                                             227   0.4  >1881.2                                                                             >5   >1881.2                                                                           >5                                             BPI.86                                                                             1520  2.58 --    --   >2048.5                                                                           >5                                             BPI.87                                                                             189   0.32 >1535.8                                                                             >5   >1535.8                                                                           >5                                             BPI.88                                                                             70.32 0.13   540.15                                                                              1  >2380.0                                                                           >5                                             BPI.89                                                                                 229.09                                                                          0.43 >1882.4                                                                             >5   >1882.4                                                                           >5                                             BPI.90                                                                                 83.11                                                                           0.16 1763  3.32 >1863.3                                                                           >5                                             BPI.91                                                                             >3843.5                                                                             >5   --    --   --  --                                             BPI.92                                                                                331.8                                                                            0.57 --    --   --  --                                             BPI.93                                                                                212.87                                                                           0.76  >980.3                                                                             >5   --  --                                             BPI.94                                                                                922.54                                                                           1.59  >922.5                                                                             >5    >922.5                                                                           >5                                             BPI.95                                                                                330.88                                                                           0.6  >1397.6                                                                             >5   --  --                                             BPI.96                                                                                378.33                                                                           0.65 >2048.5                                                                             >5   >2048.5                                                                           >5                                             BPI.97                                                                                296.58                                                                           0.53 --    --   --  --                                             BPI.98                                                                             >1626.1                                                                             >5   >1626.1                                                                             >5   >1626.1                                                                           >5                                             BPI.99                                                                                722.9                                                                            2.99 >1064.1                                                                             >5   >1064.1                                                                           >5                                             BPI.100                                                                               407.74                                                                           0.73 >2655 >5   --  --                                             BPI.101                                                                              1329.3                                                                            4.79 >1329.3                                                                             >5   >1329.3                                                                           >5                                             BPI.102                                                                            >2635.6                                                                             >5   >2635.6                                                                             >5   --  --                                             MAP.1                                                                              106   0.82    552.79                                                                           4.27  >647.5                                                                           >5                                             MAP.2                                                                               >690.9                                                                             >5    >690.9                                                                             >5    >690.5                                                                           >5                                             __________________________________________________________________________     .sup.a Amount added to well to achieve a 30 mm.sup.2 hold as determined b     PROBIT analysis as described in Examples 15 and 16.                           .sup.b No detectable activity up to 4 μg/well.                             .sup.c N.T. = not tested.                                                

It will be recognized that BPI.84 peptide was found to have bactericidalactivity against E. coli J5 bacteria that was the molar equivalent torBPI₂₃.

The results of heparin binding experiments for the BPI functional domainpeptides of the invention are shown in representative examples in FIGS.15a-15e and FIG. 16, and summarized in Table IX, wherein heparin bindingdata are expressed as affinity (nM) and capacity (ng). By plotting theamount of heparin bound versus increasing concentration of heparin, andfitting the data to standardized equations by non-linear least squaresmethods (GraFit, v.2.0, Erithacus Software, London, England), both thebinding constant (K_(d)) and capacity are calculated.

                  TABLE IX                                                        ______________________________________                                        BPI Peptide                                                                              Heparin Affinity (nM)                                                                       Heparin Capacity (ng)                                ______________________________________                                        BPI.1      no binding    no binding                                           BPI.2      346.5         203.6                                                BPI.3      780.8         264.5                                                BPI.4      335.6         80.8                                                 BPI.5      193.4         177.6                                                BPI.7      908.0         405.6                                                BPI.8      573.8         92.2                                                 BPI.9      1141.4        212.5                                                BPI.10     915.7         548.9                                                BPI.11     743.9         290.5                                                BPI.12     284.6         231.5                                                BPI.13     984.5         369.1                                                BPI.14     396.4         119.3                                                BPI.15     315.0         145.4                                                BPI.16     231.0         127.25                                               BPI.17     266.5         113.1                                                BPI.18     381.2         156.6                                                BPI.19     266.5         203.6                                                BPI.20     485.1         203.6                                                BPI.21     231.0         50.9                                                 BPI.22     315.0         135.7                                                BPI.23     693.0         285.0                                                BPI.24     165.0         427.6                                                BPI.25     346.5         107.2                                                BPI.26     231.0         113.1                                                BPI.27     203.8         119.8                                                BPI.28     266.5         156.6                                                BPI.29     427.4         463.7                                                BPI.30     592.2         499.4                                                BPI.31     252.4         205.1                                                BPI.32     339.5         217.1                                                BPI.33     492.2         230.7                                                BPI.34     518.2         205.1                                                BPI.35     1083.0        406.0                                                BPI.36     378.7         80.2                                                 BPI.37     189.3         136.7                                                BPI.38     579.1         194.3                                                BPI.39     757.3         335.6                                                BPI.40     546.9         160.5                                                BPI.41     468.8         119.1                                                BPI.42     820.4         369.1                                                BPI.43     492.3         283.9                                                BPI.44     579.1         335.6                                                BPI.45     152.6         160.7                                                BPI.46     1067.0        321.1                                                BPI.47     1911.0        576.4                                                BPI.48     1415.0        442.3                                                BPI.54     237.4         64.3                                                 BPI.55     367.6         166.1                                                BPI.56     114.6         135.5                                                BPI.58     194.0         231.2                                                BPI.59     174.9         106.7                                                BPI.60     64.8          120.3                                                BPI.61     58.3          85.2                                                 BPI.63     599.8         305.1                                                BPI.65 (ox.)                                                                             159.5         190.6                                                BPI.65 (red.)                                                                            216.0         279.6                                                BPI.66     295.7         111.6                                                BPI.67     107.8         250.4                                                BPI.69     967.1         450.8                                                BPI.70     145.2         59.2                                                 BPI.71     75.6          158.9                                                BPI.72     145.2         102.8                                                BPI.73     227.2         413.4                                                BPI.74     218.1         207.3                                                BPI.75     96.0          119.8                                                BPI.76     127.9         144.4                                                BPI.77     301.9         581.7                                                BPI.79     199.4         110.2                                                BPI.80     135.6         210.3                                                BPI.81     334.7         318.4                                                BPI.82     427.2         163.1                                                BPI.83     409.9         253.3                                                BPI.84     1003.2        329.2                                                BPI.85     682.4         233.1                                                BPI.86     383.1         208.4                                                BPI.87     575.0         280.0                                                BPI.88     1629.0        352.8                                                BPI.89     1199.4        252.8                                                BPI.90     1231.7        274.8                                                BPI.91     288.1         181.2                                                BPI.92     667.1         227.3                                                BPI.93     386.7         291.5                                                BPI.94     406.9         216.1                                                BPI.95     551.2         224.5                                                BPI.96     468.8         203.8                                                BPI.97     765.4         252.2                                                BPI.98     683.3         1678.4                                               BPI.99     9097.7        971.4                                                BPI.100    2928.9        314.0                                                BPI.101    1905.0        210.9                                                BPI.102    4607.8        535.2                                                MAP.1      936.8         459.1                                                MAP.2      785.5         391.2                                                Cecropin   395.3         242.0                                                Magainin   3174.6        453.7                                                PMB Peptide                                                                              309.42        58.01                                                LALF       1294.1        195.3                                                ______________________________________                                    

An intriguing relationship was observed among representative BPIfunctional domain peptides when a multiple regression analysis was doneusing bactericidal activity as the predicted variable and heparinbinding capacity and affinity (K_(d)) as the predictor variables. Thisanalysis revealed that only heparin binding capacity was significantlyrelated to bactericidal activity (heparin capacity, p=0.0001 and heparinaffinity, p=0.6007). In other words, the amount of heparin that a givenpeptide embodiment can bind at saturation (i.e. capacity) has asignificant relationship with bactericidal activity and not how soon agiven peptide reaches 50% saturation in the heparin titration (i.e.affinity). From the data on LPS binding competition and neutralization,it also appears that capacity is most predictive of bactericidalactivity. For examples, the results demonstrate that BPI.7, BPI.29,BPI.30, BPI.46, BPI.47, BPI.48, BPI.63, BPI.65 (reduced), BPI.69,BPI.73, BPI.58, MAP.1 and MAP.2 have extremely high heparin capacity andalso are highly bactericidal. Multiple antigenic peptides (MAP peptides)are multimeric peptides on a branching lysine core as described byPosnett and Tam, 1989, Methods in Enzymology 178: 739-746. Conversely,BPI.2, BPI.4, BPI.8, BPI.14, BPI.53 and BPI.54 have low heparin bindingcapacity and accordingly have little or no bactericidal activity.

BPI interdomain combination peptides BPI.30 (comprising domain II-domainIII peptides) and BPI.74 (comprising domain III-domain II peptides) werecompared for bactericidal activity against Gram-negative andGram-positive bacteria, and for heparin binding and capacity. Theseresults surprisingly showed that inverting the order of the peptides inthe combination changed the relative activity levels observed. Forexample, BPI.74 was found to have greatly reduced bactericidal activitycompared with BPI.30. Specifically, BPI.74 had 10-fold lowerbactericidal activity against E. coli J5 bacteria, 50-fold lowerbactericidal activity against E. coli O 111:B4 bacteria, and 3.5-foldlower bactericidal activity against S. aureus. A 2-fold reduction inheparin binding capacity and a 2-fold increase in heparin affinity, wasalso observed.

Other bactericidal and endotoxin binding proteins were examined forheparin binding activity. Cecropin A, magainin II amide, Polymyxin Bpeptide and Limulus anti-LPS factor (LALF) were assayed in the directheparin binding assay described in Example 3. The magainin II amide(Sigma, St. Louis, Mo.) exhibited the highest heparin binding capacity(437.7 ng heparin/2 μg peptide, K_(d) =3.17 μM) relative to cecropin A(Sigma, 242 ng/2 μg, K_(d) =395 nM), LALF (Assoc. of Cape Cod, WoodsHole, Mass., 195.3 ng/2 μg peptide, K_(d) =1.29 μM), and PMB peptide(Bachem Biosciences, Philadelphia, Pa., 58.0 ng/2 μg peptide, K_(d) =309mM). The magainin II amide is a substitution variant of the naturalmagainin sequence, where 3 alanines have been substituted at positions8, 13, 15. The magainin II amide is reported to have less hemolyticactivity than the natural magainin sequence.

The above results support the relationship between heparin binding, LPSbinding and bactericidal activities demonstrated by the BPI peptide dataand suggest that other LPS binding proteins will also bind to heparin.The more active bactericidal proteins, cecropin A and magainin II amide,correspondingly, have the highest heparin binding capacity of thisseries of other LPS binding proteins.

One type of BPI functional domain peptide addition variant incorporatesthe addition of D-alanine-D-alanine to either the amino- orcarboxyl-terminus of a BPI functional domain peptide. The rational forthis approach is to confer greater Gram-positive bactericidal activitywith the addition of D-alanine. The cell wall biosynthesis inGram-positive bacteria involves a transpeptidase reaction thatspecifically binds and utilizes D-alanine-D-alanine. Beta-lactamantibiotics such as the penicillins effectively inhibit this samereaction. Incorporation of D-alanine-D-alanine onto an activebactericidal peptide should target the peptide to the actively growingcell wall of Gram-positive bacteria.

In the domain II substitution series of BPI functional domain peptides,an unexpected increase was observed when Lys₉₅ was substituted byalanine (BPI.24). A subsequent phenylalanine substitution at position 95(BPI.73) resulted in improved activity compared with the alaninesubstitution species. Surprisingly, substitution at position 95 withD-Phe (BPI.76) resulted in dramatically reduced activity, to levelslower than the original peptide (BPI.2). This isomer effect demonstratesthat the interactions of this peptide is stereospecific, and impliesthat BPI.73 can adopt a more active conformation compared with BPI.76.Such stereospecificity, particularly after the phenomenon has beeninvestigated at other residues, provides an important determinant forpharmacophore development.

Peptides derived from the functional domains of BPI as defined hereinhave been utilized to determine that the hydrophobic amino acids(especially tryptophan) are most critical for optimal activity. Thisfinding was unexpected due the cationic nature of BPI. In fact, fordomain II, when a lysine is replaced by an alanine or phenylalanine, theactivity increases dramatically (BPI.24, BPI.73). Combinations offunctional domain peptides can also increase the potency of individualpeptide constructs, including combinations of the most activesubstitution peptides from the three domains.

The purity of each newly synthesized peptide was determined byanalytical reverse-phase HPLC using a VYDAC C-18 column (25 cm×4.6 mm, 5μm particle size, 30 nm pore size; Separation Group, Hesperia, Calif.).HPLC was performed using 5% acetonitrile/0.1% trifluoroacetic acid (TFA)in water as mobile phase A, and 80% acetonitrile/0.065% TFA as mobilephase B. The eluate was monitored spectrophotometrically at 220 nm. Theflow rate was 1.0 mL/min. Gradient elution conditions were selected togive optimum resolution for each peptide. Purity was expressed as thepercentage that the main peak area, contributed to the total peak area(see Table X). Purity and identity of the new synthesized peptides werealso determined by electrospray ionization mass spectrometry using a VGBiotech Bio-Q mass spectrometer. Table X presents a summary of thepurity analyses of exemplary peptides of the invention by mass,spectroscopy and HPLC.

                  TABLE X                                                         ______________________________________                                        Peptide #                                                                            Protein AA Segment                                                                             MS % Purity                                                                             HPLC % Purity                               ______________________________________                                        BPI.1  19-33            --        --                                          BPI.2  85-99            57        37.2                                        BPI.3  73-99            --        --                                          BPI.4  25-46            --        --                                          BPI.5   42-163          --        --                                          BPI.6  112-127          --        --                                          BPI.7  (90-99) × 2                                                                              69        40.9                                        BPI.8  90-99            79        --                                          BPI.9  95-99, 90-99     --        --                                          BPI.10 94-99, 90-99, 90-99 and                                                                        --        --                                                 93-99, 90-99, 90-99                                                    BPI.11 148-151, 153-161 --        --                                          BPI.12 141-169          --        --                                          BPI.13 148-161          78        69                                          BPI.13P                                                                              148-161          100       98                                          BPI.14 21-50            --        13, 3                                       BPI.15 85-99, A @ 85 (I)                                                                              66        57.6                                        BPI.16 85-99, A @ 86 (K)                                                                              --        84.1                                        BPI.17 85-99, A @ 87 (I)                                                                              86        77, 67                                      BPI.18 85-99, A @ 88 (S)                                                                              66        70                                          BPI.19 85-99, A @ 88 (G)                                                                              --        69                                          BPI.20 85-99, A @ 90 (K)                                                                              --        66                                          BPI.21 85-99, A @ 91 (W)                                                                              68        65.8                                        BPI.23 85-99, A @ 94 (Q)                                                                              --        69                                          BPI.24 85-99, A @ 95 (K)                                                                              --        67                                          BPI.25 85-99, A @ 96 (R)                                                                              --        73                                          BPI.26 85-99, A @ 97 (F)                                                                              --        73                                          BPI.27 85-99, A @ 98 (L)                                                                              --        65                                          BPI.28 85-99, A @ 99 (K)                                                                              --        80                                          BPI.29 (148-161) × 2                                                                            --        --                                          BPI.30 90-99, 148-161   --        21                                          BPI.30-P                                                                             90-99, 148-161   95        98                                          BPI.31 148-161, A @ 148 (K)                                                                           --        68                                          BPI.32 148-161, A @ 149 (S)                                                                           --        70                                          BPI.33 148-161, A @ 150 (K)                                                                           --        58                                          BPI.34 148-161, A @ 151 (V)                                                                           --        51                                          BPI.35 148-161, A @ 152 (G)                                                                           --        72                                          BPI.36 148-161, A @ 153 (W)                                                                           --        64                                          BPI.37 148-161, A @ 154 (L)                                                                           --        51                                          BPI.38 148-161, A @ 155 (I)                                                                           --        70                                          BPI.39 148-161, A @ 156 (Q)                                                                           --        53                                          BPI.40 148-161, A @ 157 (L)                                                                           --        53                                          BPI.41 148-161, A @ 158 (F)                                                                           --        63                                          BPI.42 148-161, A @ 159 (H)                                                                           --        59                                          BPI.43 148-161, A @ 160 (K)                                                                           --        53                                          BPI.44 148-161, A @ 161 (K)                                                                           --        70                                          BPI.45 85-99, A @ 94 (Q) & 95 (K)                                                                     71        46                                          BPI.46 (99-90) × 2, A @ 1st 94 (Q)                                                              67        47                                                 & 95 (K)                                                               BPI.47 (90-99) × 2, A @ 2d 94 (Q)                                                               57        34                                                 & 95 (K)                                                               BPI.48 (90-99) × 2, A @ both 94 (Q)                                                             68        33                                                 & 95 (K)                                                               BPI.54 21-35            --        --                                          BPI.55 152-172          --        --                                          BPI.56 85-99, K @ 94 (Q) & Q @                                                                        --        55                                                 95 (K)                                                                 BPI.58 Cys-85-99        49        25.7                                        BPI.59 85-99, A @ 90 (K) & 92 (K)                                                                     56        30.3                                        BPI.60 85-99, A @ 86 (K) & 99 (K)                                                                     57        78.3                                        BPI.61 85-99, F @ 91 (W)                                                                              60        59.8                                        BPI.63 85-99, 148-161   38        31.3                                        BPI.65 Rd                                                                            Cys-85-99-Cys    41        22, 34                                      BPI.65 Ox                                                                            Cys-85-99-Cys    --        --                                          BPI.66 85-99, W.sub.D @ 91 (W)                                                                        --        --                                          BPI.67 85-99, β-(1-naphthyl)-A @                                                                 65        52                                                 91                                                                     BPI.69  90-99, A @ 94 (Q) & 95                                                                        44        54, 40                                             (K)! × 3                                                         BPI.70 85-99, β-(3-pyridyl)-A @                                                                  66        54                                                 91                                                                     BPI.71 A.sub.D -A.sub.D -85-99                                                                        --        60                                          BPI.72 85-99, β-(3-pyridyl)-A @                                                                  --        52                                                 97 (F)                                                                 BPI.73 85-99, F @ 95 (K)                                                                              --        44, 39                                      BPI.74 148-161, 90-99   --        29                                          BPI.75 KKRAISFLGKKWQK   --        32                                          BPI.76 85-99, F.sub.D @ 95 (K)                                                                        --        39                                          BPI.77 85-99, W @ 95 (K)                                                                              --        38                                          BPI.79 85-99, K @ 94 (Q)                                                                              --        48                                          BPI.80 85-99, β-(1-naphthyl)-A @                                                                 --        44                                                 95 (K)                                                                 BPI.81 85-99, F @ 94 (Q)                                                                              --        33, 35                                      BPI.82 148-161, W @ 158 (F)                                                                           --        58                                          BPI.83 148-161, β(1-naphthyl)-A @                                                                --        63                                                 153 (W)                                                                BPI.84 85-99, β-(1-naphthyl) A @                                                                 --        50                                                 91 (W) & F @ 95 (K)                                                    BPI.85 148-161, L @ 152 (G)                                                                           --        74                                          BPI.86 148-161, L @ 156 (Q)                                                                           --        51                                          BPI.87 148-161, L @ 159 (H)                                                                           --        63                                          BPI.88 85-99, F @ 94 (Q) & 95 (K)                                                                     --        50                                          BPI.89 85-99, β-(1-naphthyl) A @                                                                 --        50                                                 91 (W) & F @ 94 (Q)                                                    BPI.90 85-99, β-(1-naphthyl) A @                                                                 --        63                                                 91 (W), F @ 94 (Q) & 95 (K)                                            BPI.91 148-161, F @ 156 (Q)                                                                           --        31                                          BPI.92 148-161, K @ 156 (Q)                                                                           --        50                                          BPI.93 85-99 148-161 β-(1-                                                                       --        38                                                 naphthyl) A @ 91 (W), F @                                                     95 (K)                                                                 BPI.94 148-161, F @ 159 (H)                                                                           --        59                                          BPI.95 148-161, F @ 152 (G)                                                                           --        57                                          BPI.96 148-161, F @ 161 (K)                                                                           --        60                                          BPI.97 148-161, K @ 161 (G)                                                                           --        67                                          BPI.98 90-99, β-(1-naphthyl) A @                                                                 --        31                                                 91 (W), F @ 95 (K) + 91                                                BPI.99  90-99, W @ 95 (K)! × 3                                                                  --        --                                          BPI.100                                                                              148-161, K @ 152 (G) &                                                                         --        --                                                 156 (Q)                                                                MAP.1  βAla--Nα,Nε-- Nα,Nε                                           54        multiple                                           (BPI.2)1Lys!Lys            peaks                                       MAP.2  βAla--Nα,Nε-- Nα,Nε                                           49        multiple                                           (BPI.13)1Lys!Lys           peaks                                       ______________________________________                                    

BPI.13, as well as other selected peptides, were purified using asemi-preparative reverse-phase VYDAC C-18 column (25 cm×10 mm, 10 μmparticle size, 30 nm pore size). The following gradient was used topurify BPI.13: 26.7%B to 33 %B/30 min. at a flow rate of 2.0 mL/min.BPI.13 was dissolved in mobile phase A at a concentration of 8.8 mg/mLand injected in a volume in 0.5 mL. Three separate injections were madeand the main peak from each injection was collected. The collectedmaterial was combined and evaporated to dryness using a SpeedVac.

The purity of the recovered material (which will be referred to asBPI.13P, for purified) was determined with the analytical reverse-phasesystem and gradient elution conditions described above. Based on thisanalysis, BPI.13P was 98% pure. Purity and identity of BPI.13P was alsodetermined by electrospray ionization mass spectometry using a VGBiotech Bio-Q mass spectrometer. The observed molecular mass was 1711.0(the predicted mass was 1711.1). No impurities were detected by massspectrometry. Recovery of BPI.13P was 55%, assuming that the desiredpeptide constituted 69% of the starting material.

When peptides of the invention were further purified, as describedabove, the magnitude of the tested biological activity of the peptides,e.g., BPI.13P and BPI.30P, were found to increase when chemical puritywas increased. This indicated that the observed biological activity wasdue to the peptide itself. In particular, the completely novel andunexpected antifungal activity of BPI.13 against Candida albicans (seeExample 16), with a purity of about 69%, was further increased when thepurity of the peptide preparation was increased to 98%.

EXAMPLE 20 Analysis of BPI Functional Domain Peptides using Binding andNeutralization Assays

A. LPS Binding Assays

BPI functional domain peptides were subjected to LPS binding assays.

The first of these assays was performed as described in Gazzano-Santoroet al., supra. Briefly, a suspension of E. coli strain J5 Lipid A wassonicated and diluted in methanol to a concentration of 0.2 μg/mL, andthen 50 μL aliquots were adsorbed to wells (Immulon 2 Removawell Strips,Dynatech). Following overnight incubation at 37° C., the wells wereblocked with 215 μL of a solution of D-PBS/0.1% BSA for 3 hr at 37° C.Thereafter, the blocking buffer was discarded, the wells were washedwith a solution of 0.05% Tween-20 in D-PBS (D-PBS/T) and incubatedovernight at 4° C. with 50 μL of a solution of ¹²⁵ I!-rBPI₂₃ in D-PBS/T(a total of 234,000 cpm at a specific activity of 9.9 μCi/μg) andincreasing concentrations of BPI functional domain peptides. After thisincubation, the wells were washed three times with D-PBS/T and the boundradioactivity counted using, a gamma counter. Binding to wells treatedwith D-PBS/BSA was considered non-specific background binding and wassubtracted from the total radioactivity bound in each well to yield theamount of specifically-bound radioactivity.

The results of these experiments are shown in FIGS. 17a (where theconcentration of each peptide is given in nM) and 17b (the identicalresults, with the concentration of peptide given in μg/mL). Competitionexperiments using unlabeled rBPI₂₃ are shown for comparison. Theseresults demonstrate that all of the tested peptides have some capacityto compete with rBPI₂₃ for LPS binding, to differing degrees.

This experiment was repeated, comparing the LPS binding affinity ofBPI.10 with rBPI₂₃, using twice the amount of ¹²⁵ !-rBPI₂₃ (a total of454,000 cpm, specific activity 10 μCi/μg) and in the presence or absenceof whole blood. These results are shown in FIG. 18, and demonstratethat, on a molar basis, BPI.10 is within a factor of 2 as potent asrBPI₂₃ in competing with radiolabeled rBPI₂₃ in this assay.

The experiment was repeated using peptides BPI.7, BPI.29 and BPI.30, asin the first experiment described above except that a total of 225,000cpm of ¹²⁵ I!-rBPI₂₃ was used and Lipid A was plated at a concentrationof 0.5 mg/mL. The results of this experiment are shown in FIG. 19, andshow that, on a molar basis, these peptides are 6- to 10-fold lesspotent that unlabeled rBPI₂₃ in binding Lipid A.

A second binding assay was developed, wherein radiolabeled recombinantLPS binding protein ( ¹²⁵ I!-rLBP) was used instead of radiolabeledrBPI₂₃ in competition experiments with BPI functional domain peptidesBPI.2, BPI.3, BPI.4, BPI.5, BPI.7, BPI.13, BPI.14, BPI.29, BPI.30andBPI.48. rBPI, rBPI₂₁ Δcys, and rLBP₂₅ were included in these assays ascontrols. In these experiments, Lipid A. was adsorbed to the wells at aconcentration of 0.7 μg/mL in methanol. Incubation of radiolabeled rLBP(a total of 650,000 cpm and a specific activity of 3.45 μCi/μg) wasperformed for 2.5 hr at 37° C. in the presence of BPI peptides in aseries of increasing concentrations. These results are shown in FIGS.20a and 20b. IC₅₀ values (i.e., the concentration at which Lipid Abinding of radiolabeled rLBP₂₅ is inhibited to one half the valueachieved in the absence of the peptide) are shown in accompanying TableXI.

                  TABLE XI                                                        ______________________________________                                                       IC50:                                                          Peptide          nM      μg/mL                                             ______________________________________                                        rBPI              13     0.65                                                 rBPI.sub.21 Δcys                                                                          30     0.69                                                 BPI.7            100     0.26                                                 BPI.29           130     0.44                                                 BPI.48           200     0.48                                                 BPI.30           250     0.75                                                 BPI.3            250     0.75                                                 rLBP.sub.25      600     15                                                   BPI.13           1000    1.7                                                  BPI.2            1300    2.36                                                 BPI.5            1700    4.42                                                 ______________________________________                                    

In a third binding assay, a number of BPI functional domain peptideswere tested for their ability to bind to radiolabeled LPS followingincubation with human endothelial cells (HUVEC). This assay measures theability to bind LPS once the BPI peptides are bound to HUVEC cells.HUVEC cells were incubated in the presence of various BPI peptides at aconcentration of either 1 μg/mL or 3 μg/mL for 3 hr at 4° C. of 500 μLof a solution of D-PBS/BSA. Following this incubation, the cells werewashed twice with ice-cold D-PBS/BSA and then incubated for anadditional 2.5 hr at 4° C. in 500 μL of a solution of ¹²⁵ !-RaLPS (atotal of 340,000 cpm at a specific activity of 4.6×10⁶ cpm/μg) inD-PBS/BSA. The wells were washed three times with D-PBS/BSA, solubilizedin 500 μL of 1M NaOH and the lysates counted using a gamma counter.These results, shown in FIG. 21, indicate that BPI.29 and BPI.30 retainthe capacity to bind LPS while bound to HUVEC cells.

B. LPS Neutralization Screening Assay of BPI Functional Domain Peptidesusing TNF Cellular Toxicity

A screening assay for LPS neutralization was developed using a tumornecrosis factor (TNF) cellular toxicity assay. A human monocytic cellline (THP-1; accession number TIB202, American Type Culture Collection,Rockville, Md.) grown in media supplemented with Vitamin D produce TNFupon stimulation with LPS in a dose-dependent fashion. Mouse fibroblasts(L929 cells; ATCC No.: CCL1) are sensitive to TNF-mediated cell killing,and this cell killing is also dose-dependent. Thus, the extent of cellkilling of L929 cells provides a sensitive assay for the degree of TNFinduction in THP-1 cells, which in turn is a sensitive indicator of theamount of free LPS in contact with the THP-1 cells. LPS binding andneutralization by BPI functional domain peptides or rBPI₂₃ reduces theamount of free LPS in contact with THP-1 cells, which reduces the amountof TNF produced, which in turn reduces the amount of L929 cell killingin a standardized assay. Thus, the following assay provides a sensitivemethod for assessing the LPS binding and neutralization capacity of theBPI functional domain peptides of this invention.

THP-1 cells were grown in RPMI media (GIBCO, Long Island, N.Y.)supplemented with 10% FCS and Vitamin D in spinner culture for 3 days toa density of about 150,000 cells/mL. Cells were then plated in around-bottomed 96-well culture plate at a density of 100,000 cells/welland incubated in RPMI media without Vitamin D or FCS in the presence of5 ng/mL E. coli 01113 LPS for 6 hr at 37° C. Experimental control wellsalso contained varying amounts of rBPI₂₃ or BPI functional domainpeptides, in concentrations varying from about 0.1 μg/mL to about 100μg/mL. After this incubation, the plates were centrifuged at about 600×g to pellet the cells, and 50 μL of the supernatant were added to a96-well flat bottomed culture dish prepared in parallel with 50,000 L929cells per well in 50 μL RPMI/10% FCS.

L929 cells were prepared by monolayer growth in RPMI/10% FCS media to adensity of about 1 million cells per dish, then split 1:2 on the daybefore the experiment and allowed to grow overnight to about 70%confluence on the day of the experiment. Actinomycin D was added to the70% confluent culture to a final concentration of 1 μg/mL 20 min priorto plating in 96-well plates. L929 cell plates were incubated in thepresence of the THP-1 supernatant for about 16 hr (overnight) at 37° C.under standard conditions of mammalian cell growth. To each well wasthen added 20 μL of a solution prepared by diluting 100 μL of phenazinemethylsulfonate in 2 mL CellTitre 96™AQ_(ueous) solution (Promega,Madison, Wis.), containing 3-(4,5-dimethyl)-thiozol-2-yl!-5-(3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium(inner salt). The cultures were allowed to incubate for 2-4 hr at 37° C.and then analyzed spectrophotometrically to determine the opticalabsorbance at 490 nm (A490). Experimental results were evaluatedrelative to a semilog standard curve prepared with known amounts of TNF,varying from about 10 ng/mL to about 10 mg/mL.

The results of these experiments are shown in FIGS. 22a-22h. FIG. 22ashows the relationship between A490 and TNF concentration in cultures ofL929 cells in the presence and absence of 5 ng/mL LPS. These resultsshow about the same linear relationship between A490 and concentrationof TNF whether or not LPS was present in the assay media. FIG. 22billustrates an experiment where TNF was incubated with L929 cells in thepresence of increasing amounts of heparin. These results show a constantand characteristic A490 for TNF at concentrations of 1 ng/mL and 0.1ng/mL, indicating that heparin does not affect L929 cell killing by TNF.FIG. 22c illustrates a control experiment, showing that rBPI₂₁ Δcysdecreased the amount of TNF-mediated L929 cell killing when incubated atthe indicated concentrations in cultures of THP-1 cells in the presenceof 5 ng/mL LPS. FIG. 22d shows that heparin could compete with LPS forbinding with rBPI₂₁ Δcys, by inhibiting the BPI-mediated inhibition ofLPS-stimulated TNF production by THP-1 cells, as measured by the L929cell killing assay.

FIG. 22e is a standard curve of A490 versus TNF as a measure ofTNF-mediated L929 cell killing; FIG. 22g shows the linearity of thestandard curve in a semilog plot over a TNF concentration range of aboutthree logs (about 1000-fold). FIG. 22f shows the THP-1 cell dependenceof the assay, wherein detectable amounts of TNF were most readilyproduced using about 100,000 THP-1 cells and LPS at a concentration ofat least 5 ng/mL. Finally, FIG. 22h shows that the assay was found to bedependent on THP-1 cell production of TNF in response to LPSstimulation; human histiocytic lymphoma cells (U937; ATCC No.: CRL1593)produced no detectable TNF when substituted in the assay for THP-1cells.

This assay was used to analyze LPS binding and neutralization capacityof a number of BPI functional domain peptides of the invention. Theseresults are shown in Table XII, and indicate that each of the peptidestested had the capacity to inhibit LPS-stimulated TNF production inTHP-1 cells, as measured by TNF-mediated L929 cell killing.

                  TABLE XII                                                       ______________________________________                                        Peptide       IC.sub.50 (μg/mL)                                            ______________________________________                                        rBPI.sub.21 Δcys                                                                      0.2                                                             BPI.7         30                                                              BPI.13        20                                                              BPI.29        2-3                                                             BPI.30        6-7                                                             BPI.48         1                                                              ______________________________________                                    

C. LPS Neutralization Screening Assay of BPI Functional Domain Peptidesusing a Cellular NO Production Assay

An additional LPS neutralization screening assay for BPI functionaldomain peptides was developed using an assay for NO production in mousecells treated with LPS (see Lorsbach et al., 1993, J. Biol. Chem. 268:1908-1913). In this, assay, mouse RAW 264.7 cells (ATCC Accession No.T1B71) were treated with bacterial LPS. The cells were incubated in96-well plates and stimulated for 2 hours with E. coli O 113 LPS orzymosan, in the presence or absence of γ-interferon, rLBP, fetal bovineserum (FBS) or normal human serum (NHS), or rBPI₂₁ Δcys. After thisincubation, the cells were washed with fresh media and incubatedovernight in media containing 10% FCS. The NO released from the cellsaccumulated in the media and spontaneously converted to nitrite. Thisnitrite was assayed in situ by the Griess reaction, as follows. Thenitrite was reacted with the primary amine of an added sulfanilamide andformed a diazonium salt. This salt was then reacted with addednaphthylethylenediamine to form a red azo-dye. The Griess reaction wasperformed at room temperature in about 10 minutes. The amount ofproduced NO was estimated from a standard curve of Griess reactionproducts determined spectrophotometrically as Absorbance at a wavelengthof 550 nm.

The results of this assay are shown in FIGS. 23a to 23c. FIG. 23a showsthe dependence of NO production on the presence of γ-interferon. Thisinterferon effect was found to saturate at a concentration of 100 U/mL.FIG. 23b shows the dependence of LPS-stimulated NO production on thepresence of LBP, either added as purified recombinant protein or as acomponent of PBS or NHS supplements of the cell incubation media. FIG.23c shows rBPI₂₃ -mediated inhibition of LPS-stimulated NO production,having an IC₅₀ of 30-100 ng/mL. These results demonstrated that thisassay is a simple, inexpensive and physiologically-relevant assay systemfor assessing the LPS-neutralizing activity of BPI and BPI functionaldomain peptides disclosed herein.

The results of such assays performed with BPI functional domain peptidesare shown in FIGS. 24a-24g wherein the background production of NO byunstimulated cells is designated as "NO LPS". FIGS. 24a and 24b showinhibition of NO production stimulated by zymosan and LPS, respectively,by rBPI, rBPI₂₁ Δcys and rLBP₂₅. No inhibition of zymosan-stimulated NOproduction was seen at any concentration of BPI protein (FIG. 24a). Incontrast, LPS-stimulated NO production was inhibited in aconcentration-dependent manner by incubation with these rBPI-relatedproteins (FIG. 24b). FIG. 24c (zymosan) and FIG. 24d (LPS) shows theeffects on NO production by RAW 264.7 cells of incubation with BPI.2,BPI.3, BPI.4, BPI.7 and BPI.14; rBPI₂₁ Δcys is also shown forcomparison. As shown with native BPI, zymosan-stimulated NO productionwas not inhibited by incubation with any of the BPI functional domainpeptides (with the possible exception of a small amount of inhibition byBPI.7 at high concentrations; FIG. 24c). LPS-stimulated NO production,on the other hand, was inhibited efficiently by rBPI₂₁ Δcys, and to alesser degree by BPI.3 and BPI.7 (FIG. 24d).

This experiment was repeated using BPI.5, BPI.13, BPI.29 and BPI.30,with rBPI₂₁ Δcys analyzed in parallel for comparison. Zymosan-stimulatedNO production by RAW 264.7 cells was found to be inhibited by BPI.30 athigh (˜100 μg/mL) concentrations; neither any of the other BPIfunctional domain peptides nor rBPI₂₁ Δcys showed any inhibition ofzymosan-stimulated NO production (FIG. 24e). LPS-stimulated NOproduction was inhibited efficiently by rBPI₂₁ Δcys, and to varying andlesser degrees by all of the BPI functional domain peptides tested inthis experiment (FIG. 24f).

The IC₅₀ values (i.e., the concentration of inhibitor at which zymosanor LPS-stimulated NO production by RAW 264.7 cells is reduced toone-half its value in the absence of the inhibitor) for the BPI proteinsand peptides were calculated from these experiments and are showed inFIG. 24g. With the exception of BPI.30, no significant inhibition ofzymosan-mediated NO production was found for either the BPI functionaldomain peptides or rBPI₂₁ Δcys, rBPI or rLBP in these experiments; theIC₅₀ of BPI.30 for inhibition of zymosan-stimulated NO production wasfound to be between 10 and 100 μg/mL. BPI.3, BPI.5, BPI.13, BPI.29 andBPI.30 were found to have detectable levels of LPS neutralization inthis assay, and the relative IC₅₀ values for these peptides are shown inFIG. 24g.

D. LPS Neutralization Screening Assay of BPI Functional Domain Peptidesusing a Cellular Proliferation Assay

An additional LPS neutralization screening assay for evaluation of BPIfunctional domain peptides was developed. This sensitive assay forinhibition of cellular proliferation in mouse cells treated with LPS canalso be utilized for quantitation of LPS levels in human plasma upondevelopment of a standard curve.

In this assay, mouse RAW 264.7 cells (ATCC Accession No. T1B71),maintained in RPMI 1640 media (GIBCO), supplemented with 10 mM HEPESbuffer (pH 7.4), 2 mM L-glutamine, penicillin (100 U/mL), streptomcin(100 μg/mL), 0.075% sodium bicarbonate, 0.15M 2-mercaptoethanol and 10%fetal bovine serum (Hyclone, Inc., Logan, Utah), were first induced byincubation in the presence of 50 U/mL recombinant mouse γ-interferon(Genzyme, Cambridge, Mass.) for 24 h prior to assay. Induced cells werethen mechanically collected and centrifuged at 500 ×g at 4° C. and thenresuspended in 50 mL RPMI 1640 media (without supplements),re-centrifuged and again resuspended in RPMI 1640 media (withoutsupplements). The cells were counted and their concentration adjusted to2×10⁵ cells/mL and 100 μL aliquots were added to each well of a 96-wellmicrotitre plate. The cells were then incubated for about 15 hours withE. coli O 113 LPS (Control Standard, Assoc. of Cape Cod, Woods Hole,Mass.), which was added in 100 μL/well aliquots at a concentration of 1ng/mL in serum-free RPMI 1640 media (this concentration being the resultof titration experiments in which LPS concentration was varied between50 pg/mL and 100 ng/mL). This incubation was performed in the absence orpresence of BPI functional domain peptides in varying concentrationsbetween 25 ng/mL and 50 μg/mL. Recombinant human BPI was used as apositive control at a concentration of 1 μg/mL. Cell proliferation wasquantitatively measured by the addition of 1 μCi/well ³ H!-thymidine 5hours after the time of initiation of the assay. After the 15-hourincubation, labeled cells were harvested onto glass fiber filters with acell harvester (Inotech Biosystems, INB-384, Sample Processing andFilter Counting System, Lansing, Mich.).

The results of this assay are shown in FIGS. 26a-26c. FIG. 26a shows thedependence of LPS-mediated inhibition of RAW 264.7 cell proliferation ofthe presence of LBP, added to the reaction mixture either as a componentof serum or as recombinant LBP (at a concentration of 1 μg/mL). FIGS.26b and 26c illustrate patterns of BPI functional domain peptidebehavior found in the above assay. BPI.5 displayed an EC₅₀ (i.e., thepeptide concentration at which the growth inhibitory effect of LPS wasreversed by 50%) of 5.3±0.6 μg/mL. BPI.81 was unable to reverse thegrowth inhibitory effect of LPS on RAW 264.7 cells, but showedadditional growth inhibition with an IC₅₀ (i.e., the peptideconcentration at which RAW cell growth was inhibited by 50% from thevalue without added peptide) of 14±0.2 g/mL. BPI.98 showed an EC₅₀ of0.16±0.08 μg/mL and an IC₅₀ of 16.5±1.9 μg/mL. Finally, BPI.86 showed anEC₅₀ of 0.13±0.04 μg/mL and an IC₅₀ of 37.5±12.5 μg/mL. Results from allpeptides tested with this assay are shown in Table XIII.

                                      TABLE XIII                                  __________________________________________________________________________    BPI peptide                                                                         Sequence                EC.sub.50                                                                           IC.sub.50                                 __________________________________________________________________________    BPI.2 IKISGKWKAQKRFLK         --    --                                        BPI.5 HVHISKSKVGWLIQLFHKKIE   5.3 ± 0.6                                                                        --                                        BPI.7 KWKAQKRFLKKWKAQKRFLK    >50   37.5 ± 12.5                            BPI.10                                                                              QKRFLKKWKAQKRFLKKWKAQKRFLK                                                                            >50   17.25                                     BPI.13                                                                              KSKVGWLIQLFHKK          1.9 ± 0.4                                                                        37.5 ± 12.5                            BPI.13p                                                                             KSKVGWLIQLFHKK          2.0 ± 0.3                                                                        >50                                       BPI.29                                                                              KSKVGWLIQLFHKKKSKVGWLIQLFHKK                                                                           0.1 ± 0.02                                                                      13.6 ± 0.4                             BPI.30                                                                              KWKAQKRFLKKSKVGWLIQLFHKK                                                                              1.2 ± 1.1                                                                        10.5 ± 1.2                             BPI.46                                                                              KWKAAAKRFLKKWKAQKRFLK   1.9 ± 1.9                                                                        18.8 ± 0.8                             BPI.47                                                                              KWKAQKRFLKKWKAAAKRFLK   0.9 ± 0.3                                                                        9.8 ± 0.1                              BPI.48                                                                              KWKAAAKRFLKKWKAAAKRFL   1.3 ± 0.9                                                                        5.0 ± 0.1                              BPI.63                                                                              IKISGKWKAQKRFLKKSKVGWLIQLFHKK                                                                         0.08 ± 0.02                                                                       7.1 ± 0.02                            BPI.69                                                                              KWKAAARFLKKWKAAARFLKKWKAAARFLK                                                                        0.11 ± 0.07                                                                      2.4 ± 0.3                              BPI.73                                                                              IKISGKWKAQFRFLK         22 ± 10                                                                          --                                        BPI.74                                                                              KSKVGWLIQLFHKKKWKAQKRFLK                                                                              2.7 ± 0.3                                                                        18.8 ± 0.8                             BPI.76                                                                              IKISGKWKAQF.sub.D RFLK  >50   --                                        BPI.77                                                                              IKISGKWKAQWRFLK         10 ± 32                                                                          >50                                       BPI.80                                                                              IKISGKWKAQA.sub.β-(1-naphthyl) RFLK                                                              35 ± 36                                                                          >50                                       BPI.81                                                                              IKISGKWKAFKRFLK         --    14.0 ± 0.2                             BPI.82                                                                              KSKVGWLIQLWHKK          0.8 ± 0.1                                                                        18.8 ± 0.8                             BPI.83                                                                              KSKVGWLIQLA.sub.β-(1-naphthyl) HKK                                                               1.2 ± 0.1                                                                        37.5 ± 12.5                            BPI.84                                                                              IKISGKA.sub.β-(1-naphthyl) KAQFRFLK                                                              57 ± 28                                                                          --                                        BPI.85                                                                              KSKVLWLIQLFHKK          1.3 ± 0.1                                                                        17 ± 15                                BPI.86                                                                              KSKVGWLILLFHKK          0.13 ± 0.04                                                                      37.5 ± 12.5                            BPI.87                                                                              KSKVGWLIQLFLKK          1.3 ± 0.4                                                                        11.4 ± 1.3                             BPI.88                                                                              IKISGKWKAFFRFLK         >50   6.2 ± 7.5                              BPI.89                                                                              IKISGKAβ-(1-naphthyl)KAFKRFLK                                                                    >50    11 ± 0.3                              BPI.90                                                                              IKISGKA.sub.β-(1-naphthyl) KAFFRFLK                                                              >50   6.3 ± 0.7                              BPI.91                                                                              KSKVGWLIFLFHKK          0.7 ± 0.1                                                                        --                                        BPI.92                                                                              KSKVGWLIKLFHKK          1.9 ± 0.1                                                                        37.5 ± 12.5                            BPI.93                                                                              IKISGKA.sub.β-(1-naphthyl) KAQFRFLKKSKVGWLIQLFHKK                                                 0.9 ± 0.25                                                                      9.7 ± 0.1                              BPI.94                                                                              KSKVGWLIQLFFKK           1.3 ± 0.02                                                                      23 ± 2                                 BPI.95                                                                              KSKVFWLIQLFHKK           1.0 ± 0.01                                                                      37.5 ± 12.5                            BPI.96                                                                              KSKVGWLIQLFHKF          1.6 ± 0.2                                                                        18.8 ± 0.8                             BPI.97                                                                              KSKVKWLIQLFHKK          2.8 ± 0.3                                                                        37.5 ± 12.5                            BPI.98                                                                              IKISGKA.sub.β-(1-naphthyl) KAQFRFLKKSKVGWLIFLFHKK                                                0.16 ± 0.08                                                                      16.5 ± 1.9                             MAP.1 (β-alanyl-Nα,Nε-substituted- Nα,Nε(          BPI.2)lysyl!lysine)     0.45 ± 0.1                                                                       37.5 ± 12.5                            rBPI.sub.21 Δcys        0.08 ± 0.05                                                                      --                                        __________________________________________________________________________     -- No proliferation decrease up to 50 μg/ml.                          

E. LPS Neutralization Assay based on Inhibition of LPS-induced TNFProduction in Whole Blood

LPS neutralization by BPI functional domain peptides of the inventionwas assayed in whole blood as follows. Freshly drawn blood from healthyhuman donors was collected into vacutainer tubes (ACD, Rutherford, N.J.)Aliquots of blood (170 μL) were mixed with 10 μL Ca⁺⁺ -, Mg⁺⁺ -free PBScontaining 2.5 ng/mL E. coli 0113 LPS, and with 20 μL of varyingconcentrations of the BPI peptides of the invention ranging inconcentration from 0.5-50 μg/mL. These mixtures were then incubated for4 h at 37° C., and then the reaction stopped by the addition of 55 μLice-cold Ca⁺⁺ -, Mg⁺⁺ -free PBS, followed by centrifugation at 500 ×gfor 7 min. Supernatants were then assayed for TNF levels using acommercial ELISA kit (Biokine™ ELISA Test, T-cell Sciences, Cambridge,Mass.).

The results of these experiments with representative peptides usingwhole blood samples from two different donors are shown in FIGS. 27A,27B and Table XIV. FIGS. 27A and 27B show a comparison of TNF inhibitionby BPI functional domain peptides BPI.7, BPI.13 and BPI.29; resultsobtained using rBPI₂₁ Δcys are shown for comparison. These results arequantitated as IC₅₀ values in Table XIV, and compared with LPSneutralization as assayed using NO production by RAW 264.7 cells asdescribed in Section C above.

                  TABLE XIV                                                       ______________________________________                                                   IC.sub.50 (μg/ml)                                               BPI Peptide  TNF assay  NO assay                                              ______________________________________                                        rBPI.sub.21 Δ.sub.cys                                                                0.65       0.4                                                   BPI.29       5.0        2.4                                                   BPI.13       42         16                                                    BPI.7        Not Inhibitory                                                                           Not Inhibitory                                        ______________________________________                                    

F. LPS and Heparin Binding Assays using Tryptophan FluorescenceQuenching

The naturally-occurring amino acid tryptophan can emit light (i.e., itfluoresces) having a wavelength between 300 and 400 nm after excitationwith light having a wavelength of between about 280 nm and 290 nm,preferably 285 nm. The amount of emitted light produced by suchfluorescence is known to be affected by the local environment, includingpH and buffer conditions, as well as binding interactions betweenproteins and other molecules. Some BPI functional domain peptidesderived from domains II and III contain tryptophan residues, andtryptophan fluorescence was used to assay binding interactions betweenthe BPI functional domain peptides of the invention and LPS or heparin.

Tryptophan fluorescence of the BPI functional domain peptides of theinvention was determined in the presence or absence of LPS or heparinusing a SPEX Fluorolog fluorimeter. Samples were excited with 285 nmlight using a 0.25 nm slitwidth. Emission wavelengths were scannedbetween 300-400 nm using a 1.25 nm slitwidth. Data were accumulated asthe average of three determinations performed over an approximately 5min time span. Samples were maintained at 25° C. or 37° C. during thecourse of the experiments using a circulating water bath. Crab endotoxinbinding protein (CEBP), a protein wherein the intrinsic fluorescence oftryptophan residues is affected by binding to LPS, was used as apositive control. (See Wainwright et al., 1990, Cellular and MolecularAspects of Endotoxin Reactions, Nowotny et al., eds., Elsevier SciencePublishing B.V., The Netherlands, pp. 315-325).

The results of these experiments are shown in Table XV. K_(d) valueswere determined by Scatchard-type Stern-Volmer plots of the quenchingdata as the negative inverse of the slope of such plots. Comparing thedata for BPI.10, BPI.46 and BPI.47, it is seen that as the K_(d)decreased (indicating an increase in avidity for LPS), the percentfluorescence quenching increased. The differences between these peptidesinclude replacement of basic and polar amino acid residues withnon-polar residues in BPI.48 as compared with BPI.10. In contrast, asthe K_(d) of heparin binding decreased, a corresponding increase in thepercentage of fluorescence quenching was not detected. This result mayindicate fundamental differences between the site or nature of heparinbinding compared with LPS binding.

                  TABLE XV                                                        ______________________________________                                        BPI            K.sub.d  Quenching                                                                            K.sub.d Heparin                                                                       Quenching                              Peptide                                                                              # of Trp                                                                              LPS (nM) LPS (%)                                                                              (μM) Heparin (%)                            ______________________________________                                        BPI.10 2       124      26     1.2     67                                     BPI.47 2       115      41     2.2     47                                     BPI.48 2       83       62     0.8     41                                     BPI.69 3       58       72     0.4     42                                     BPI.73 1       66       47     0.7     19                                     CEBP.sup.a                                                                           5       19       56     0.8     54                                     ______________________________________                                         .sup.a CEBP (LALF) experiments were performed at 25° C.           

G. Neutralization Assay Of Heparin-Mediated Lengthening of Thrombin Time

The effect of BPI functional domain peptides on heparin-mediatedlengthening of thrombin time, i.e., the time required for clotting of amixture of thrombin and plasma, was examined. Thrombin time islengthened by the presence of endogenous or exogenous inhibitors ofthrombin formation, such as therapeutically administered heparin. Agentswhich neutralize the anti-coagulant effects of heparin will reduce thethrombin time measured by the test.

In these experiments, thrombin clotting time was determined using a MLAElectra 800 Coagulation Timer. Reconstituted plasma (200 μL, SigmaChemical Co., No. 855-10) was incubated at 37° C. for two minutes in areaction cuvette. Thrombin Clotting Time reagent (100 μL, BaxterDiagnostics Inc., B4233-50) was added to the reaction cuvette afterincubation and clotting time was then measured. Heparin sodium (13 μL,40 μg/mL in PBS, Sigma Chemical Co., H3393) and exemplary BPI functionaldomain peptides (10 μL of various dilutions from about 0.05 μg/ml toabout 10 μg/ml) were added to the reaction cuvette prior to plasmaaddition for testing of the effects of these peptides on thrombinclotting time. TCT clotting time (thrombin time) was measured using theBPI peptides indicates and the results are shown in FIG. 28 and TableXVI. These results shown in FIG. 28 and Table XVI below demonstrate thatthe tested BPI functional domain peptides neutralized heparin, as shownby inhibition of the heparin-mediated lengthening of thrombin time. TheIC₅₀ of this inhibition was quantitated and is shown in Table XVI.

                  TABLE XVI                                                       ______________________________________                                        BPI Peptide   IC.sub.50 (μg/ml) ± SE                                    ______________________________________                                        BPI.10        0.115 ± 0.014                                                BPI.47        0.347 ± 0.041                                                BPI.63        0.362 ± 0.034                                                BPI.69        0.200 ± 0.025                                                BPI.73        0.910 ± 0.821                                                BPI.82        0.200 ± 0.073                                                BPI.84        0.225 ± 0.029                                                BPI.87        0.262 ± 0.009                                                BPI.88        0.691 ± 0.180                                                BPI.90        0.753 ± 0.210                                                BPI.98        0.242 ± 0.038                                                BPI.99        0.273 ± 0.011                                                BPI.100       0.353 ± 0.050                                                BPI.101       0.285 ± 0.088                                                BPI.102       0.135 ± 0.024                                                ______________________________________                                    

EXAMPLE 21

Heparin Neutralization Assay Based on Inhibition of Heparin/FGF-InducedAngiogenesis into Matrigel® Basement Membrane Matrix In Vivo

BPI functional domain peptides of the invention are assayed for theirability to inhibit heparin-induced angiogenesis in vivo in mice. LiquidMatrigel® (Collaborative Biomedical Products, Inc., Bedford, Mass.) ismaintained at 4° C. and angiogenic factors are added to the gel in theliquid state as described in Passaniti et al. (1992, Lab. Invest. 67:519-528). Heparin (Sigma, St. Louis, Mo.) is dissolved in sterile PBS tovarious concentrations ranging from 1,250-10,000 U/mL. Recombinantfibroblast growth factor (bhFGF; BACHEM Bioscience Inc., Philadelphia,Pa.) is diluted to 200 ng/mL with sterile PBS. A volume of 2.5 μLdissolved heparin solution and 2.5 μL recombinant bhFGF is added to 0.5mL Matrigel® per mouse injection. BPI functional domain peptides areadded to this Matrigel® mixture at varying concentrations ranging from0.5 to 50 μg/mL (final concentration) in 10 μL/0.5 mL Matrigel® aliquotper experimental animal. Ten μL sterile PBS is substituted for BPIfunctional domain peptides in Matrigel® aliquots injected into controlanimals.

Male C57BL/6J mice (Jackson Laboratory, Bar Harbor, Me.) at 6-8 weeks ofage are injected subcutaneously down the dorsal midline with 0.5 mLaliquots of Matrigel® prepared as described above. Seven days afterinjection, the Matrigel® gels are excised and placed in 500 μL Drabkin'sreagent (Sigma). Total protein and hemoglobin content are determined forthe gels stored in Drabkin's reagent after mechanical homogenization ofthe gels. Total protein levels are determined using a microplate assaythat is commercially embodied in a kit (DC Protein Assay, BioRad,Richmond, Calif.). Hemoglobin concentration is measured using SigmaProcedure #525 and reagents supplied by Sigma (St. Louis, Mo.) to beused with this procedure. Hemoglobin levels are expressed relative tototal protein concentration.

Gels to be used for histological staining are formalin-fixed immediatelyafter excision from the animals rather than being placed in Drabkin'sreagent. Formalin-fixed gels are embedded in Tissue-Tek O.C.T. compound(Miles, Inc., Elkhart, Ind.) for frozen sectioning. Slides of frozensections are stained with hematoxylin and eosin (as described byHumason, 1979, Animal Tissue Techniques, 4th Ed. W. H. feeman & Co., SanFransisco, Calif., Ch.9, pp 111-131)

The effect of the BPI functional domain peptides of the invention aredetected by microscopic examination of frozen stained sections forinhibition of angiogenesis relative to Matrigel® gel slices preparedwithout added BPI peptides. The extent of angiogenesis inhibition isquantitated using the normalized amounts of hemoglobin found in BPIpeptide-containing gel slices.

EXAMPLE 22

Analysis of BPI Functional Domain Peptides in Chronic InflammatoryDisease: Collagen-Induced or Reactive Arthritis Models

BPI functional domain peptides are administered for their effects in acollagen-induced arthritis model. Specifically, arthritis is induced inmice by intradermal immunization of bovine Type II collagen at the baseof the tail according to the method of Stuart et al. (1982, J. Clin.Invest. 69: 673-683). Generally, mice begin to develop arthriticsymptoms at day 21 after collagen immunization. The arthritic scores ofthe treated mice are then evaluated in a blinded fashion over a periodof 120 days for mice treated on each of days 21-25 with doses of eitherBPI functional domain peptides, control rBPI₂₃ or rBPI, or buffer whichare injected intravenously via the tail vein.

Specifically, bovine Type II collagen (Southern BiotechnologyAssociates, Inc., Birmingham Ala.) is administered via intradermalinjection (0.1 mg/mouse) at the base of the tail on day 0 to groups ofmale mice (Mouse/DBA/1J), each weighing approximately 20-25 g. BPIfunctional domain peptides, and rBPI₂₃ and rBPI are dissolved in abuffer comprised of 0.5M NaCl, 20 mM sodium acetate (pH 6.0) and dilutedwith PBS buffer for administration at various concentrations. PBS bufferalone (0.1 mL) is administered as a control.

The collagen-induced arthritis model is also used to evaluate theperformance of BPI functional domain peptides in comparison withprotamine sulfate. Specifically, BPI peptides are dissolved in PBS asdescribed above and administered at various concentrations. The othertest materials are administered at the following dosages: protaminesulfate (Sigma Chemical Co., St. Louis, Mo.) (0.13 mg/mouse), thaumatin(0.12 mg/mouse), and PBS buffer (0.1 mL). Groups of mice receive test orcontrol materials through intravenous injection via the tail vein oneach of days 28 through 32 post-injection with collagen.

BPI functional domain peptides are also administered to treat reactivearthritis in a Yersinia enterocolitica reactive arthritis modelaccording to the method of Yong et al. (1988, Microbial Pathogenesis 4:305-310). Specifically, BPI peptides are administered to DBA/2J micewhich have previously been injected intravenously with Yersiniaenterocolitica cWA 0:8 T2 (i.e., lacking the virulence plasmid accordingto Yong et al., supra) at a dosage of 4×10⁸ bacteria calculated toinduce a non-septic arthritis in the mice. Groups of mice each receivetest or control materials through intravenous injection via the tailvein.

Borrelia burgdorferi is the pathogen responsible for Lyme Disease andassociated arthritis and it possesses an LPS-like complex on its cellwalls which is different from but structurally related to that of E.coli. The effect of administration of BPI functional domain peptides oninhibition of B. burgdorferi LPS in a Limulus Amoebocyte Lysate (LAL)inhibition assay is determined. Specifically, an LAL assay according tothe method of Example 4 is conducted measuring the effect of BPIpeptides on B. burgdorferi LPS administered at 2.5 μg/mL and E. coli0113 LPS administered at 2 ng/mL.

EXAMPLE 23

Analysis of BPI Functional Domain Peptides in Mouse Malignant MelanomaCell Metastasis Model

BPI functional domain peptides, protamine, or buffer controls areadministered to test their efficacy in a mouse malignant melanomametastasis model. Specifically, groups of C57BL/6J mice are inoculatedwith 10⁵ B16.F10 malignant melanoma cells via intravenous injection intothe tail vein on day 0. BPI functional domain peptides in variousconcentrations are administered into the tail vein of test mice on days1, 3, 6, 8, 10, 13, 15, 17, and 19. Protamine sulfate (0.13 mg/mouse) asa positive control, or PBS buffer (0.1 mL/mouse) as a negative controlare similarly administered to additional groups of control mice. Theanimals are sacrificed via cervical dislocation on day 20 forobservation of lung tissues. The lobes of each lung are perfused andinflated by injecting 3 mL water into the lung via the trachea.Superficial tumor nodules are then counted with the aid of a dissectingmicroscope and the number of tumors found per group analyzed forstatistically significant differences.

EXAMPLE 24

Analysis of BPI Functional Domain Peptides in a Mouse Cerebral CapillaryEndothelial Cell Proliferation Assay

BPI functional domain peptides are tested for their effects in allendothelial cell proliferation assay. For these experiments, murinecerebral capillary endothelial cells (EC) as described in Bauer (1989,Microvascular Research 37: 148-161) are passaged in Medium 199containing Earle's salts, L-glutamine and 2.2 g/L of sodium bicarbonate(GIBCO, Grand Island, N.Y.), plus 10% heat inactivated fetal calf serum(FCS; Irvine Scientific, Irvine, Calif.) and 1% penicillin/streptomycin(GIBCO). Harvesting of the confluent cells is performed bytrypsinization with trypsin-EDTA (GIBCO) for 3 minutes. Trypsinizationis stopped by adding 10 mL of the passage medium to the flask.Proliferation assays are performed on freshly harvested EC in standardflat bottom 96-well microtiter plates. A final volume of 200 μL/well ismaintained for each well of the assay. A total of 4×10⁴ EC cells isadded to each well with varying concentrations of BPI peptides, orbuffer control. After 48 hours of culture in a 5% CO₂ incubator, 1 μCiof ³ H!thymidine in 10 μL of Medium 199 is added to each well. After a24 hour pulse, the EC cells are harvested by trypsinization onto glassmicrofiber filters and incorporated ³ H!thymidine is quantitated with agas proportional solid phase beta counter.

Direct binding studies of BPI peptides on EC cells are performed byharvesting the 10-times passaged cells from a confluent flask andresuspending the trypsinized cells in 12.5 mL of culture medium. Then,0.5 mL of the cell suspension is added to each well of a standard 24well tissue culture plate and incubated overnight. The plate is washedwith 0.1% bovine serum albumin in phosphate buffered saline containingcalcium and magnesium (GIBCO). After washing, 0.5 mL BSA/PBS is addedper well. Concentration dependent inhibition of EC cell proliferation ismeasured in terms of decreases in ³ H!-thymidine uptake.

EXAMPLE 25 Analysis of BPI Function Domain Peptides in Animal Models

A. Analysis in a Mouse Endotoxemia Model

BPI functional domain peptides are tested for their efficacy in a mouseexperimental endotoxemia model. Groups of at least 15 mice areadministered at intravenous injection of endotoxin (e.g., E. coli O111:B4, Sigma Chemical Co., St. Louis, Mo.) at a LD₉₀ dosage (e.g., 40mg/kg). This is followed by a second intravenous injection of the testpeptide in varying concentrations from about 0.1 mg/kg to about 100mg/kg, preferably in the range of about 1 to 50 mg/kg. Injections ofbuffer without added peptide are used in negative control mice. Theanimals are observed for 7 days and mortality recorded. The efficacy ofthe peptides of this invention is measured by a decrease inendotoxemia-associated mortality in peptide-injected mice as comparedwith control mice.

B. Analysis in a Mouse Peritonitis Model

BPI functional domain peptides are tested for their efficacy in a mousemodel of acute peritonitis. Groups of at least 15 mice are challengedwith 10⁷ live E. coli bacteria strain 07:K1 in 0.5 mL and then treatedwith 1.0 mL of a solution of BPI functional domain peptides at varyingconcentrations from about 0.1 mg/kg to about 100 mg/kg. Injections ofbuffer without added peptide are used in negative control mice. Theanimals are observed for 7 days and mortality recorded. Effective BPIfunctional domain peptides show a decrease in mortality of test groupmice compared with control group mice.

EXAMPLE 26 Therapeutic Use of BPI Functional Domain Peptides in a HumanIn vivo Endotoxin Neutralization Model

A controlled, double-blind crossover study is designed and conducted asin co-owned, copending U.S. patent application Ser. No. 08/188,221 filedJan. 24, 1994, to investigate the effects of BPI functional domainpeptides in humans rendered endotoxemic by intravenous infusion ofbacterial endotoxin.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 98                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "Domain I"                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AlaSerGlnGlnGlyThrAlaAlaLeuGlnLysGluLeuLysArgIle                              151015                                                                        LysIleProAspTyrSerAspSerPheLysIleLysHis                                       2025                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.14"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GlyThrAlaAlaLeuGlnLysGluLeuLysArgIleLysIleProAsp                              151015                                                                        TyrSerAspSerPheLysIleLysHisLeuGlyLysGlyHis                                    202530                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.4"                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       LeuGlnLysGluLeuLysArgIleLysIleProAspTyrSerAspSer                              151015                                                                        PheLysIleLysHisLeu                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.1"                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GlnGlnGlyThrAlaAlaLeuGlnLysGluLeuLysArgIleLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.54"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GlyThrAlaAlaLeuGlnLysGluLeuLysArgIleLysIlePro                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "Domain II"                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       SerSerGlnIleSerMetValProAsnValGlyLeuLysPheSerIle                              151015                                                                        SerAsnAlaAsnIleLysIleGlyLysTrpLysAlaGlnLysLysArg                              202530                                                                        PheLeuLys                                                                     35                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.2"                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       IleLysIleSerGlyLysTrpLysAlaGlnLysArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.8"                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       LysTrpLysAlaGlnLysArgPheLeuLys                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.58"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CysIleLysIleSerGlyLysTrpLysAlaGlnLysArgPheLeu                                 151015                                                                        Lys                                                                           (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.65 oxidized"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CysIleLysIleSerGlyLysTrpLysAlaGlnLysArgPheLeu                                 151015                                                                        LysCys                                                                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.3"                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      AsnValGlyLeuLysPheSerIleSerAsnAlaAsnIleLysIleSer                              151015                                                                        GlyLysTrpLysAlaGlnLysArgPheLeuLys                                             2025                                                                          (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "Domain III"                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ValHisValHisIleSerLysSerLysValGlyTrpLeuIleGlnLeu                              151015                                                                        PheHisLysLysIleGluSerAlaLeuArgAsnLys                                          2025                                                                          (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.11"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      LysSerLysValTrpLeuIleGlnLeuPheHisLysLys                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.12"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      SerValHisValHisIleSerLysSerLysValGlyTrpLeuIleGln                              151015                                                                        LeuPheHisLysLys                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.13"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      LysSerLysValGlyTrpLeuIleGlnLeuPheHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.15"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AlaLysIleSerGlyLysTrpLysAlaGlnLysArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.16"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      IleAlaIleSerGlyLysTrpLysAlaGlnLysArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.17"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      IleLysAlaSerGlyLysTrpLysAlaGlnLysArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.18"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      IleLysIleAlaGlyLysTrpLysAlaGlnLysArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.19"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      IleLysIleSerAlaLysTrpLysAlaGlnLysArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.20"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      IleLysIleSerGlyAlaTrpLysAlaGlnLysArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.21"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      IleLysIleSerGlyLysAlaLysAlaGlnLysArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.22"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      IleLysIleSerGlyLysTrpAlaAlaGlnLysArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.23"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      IleLysIleSerGlyLysTrpLysAlaAlaLysArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.24"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      IleLysIleSerGlyLysTrpLysAlaGlnAlaArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.25"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      IleLysIleSerGlyLysTrpLysAlaGlnLysAlaPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.26"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      IleLysIleSerGlyLysTrpLysAlaGlnLysArgAlaLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.27"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      IleLysIleSerGlyLysTrpLysAlaGlnLysArgPheAlaLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.28"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      IleLysIleSerGlyLysTrpLysAlaGlnLysArgPheLeuAla                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.59"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      IleLysIleSerGlyAlaTrpAlaAlaGlnLysArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.45"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      IleLysIleSerGlyLysTrpLysAlaAlaAlaArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.60"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      IleAlaIleSerGlyLysTrpLysAlaGlnLysArgPheLeuAla                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.31"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      AlaSerLysValGlyTrpLeuIleGlnLeuPheHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.32"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      LysAlaLysValGlyTrpLeuIleGlnLeuPheHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.33"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      LysSerAlaValGlyTrpLeuIleGlnLeuPheHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.34"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      LysSerLysAlaGlyTrpLeuIleGlnLeuPheHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.35"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      LysSerLysValAlaTrpLeuIleGlnLeuPheHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.36"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      LysSerLysValGlyAlaLeuIleGlnLeuPheHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.37"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      LysSerLysValGlyTrpAlaIleGlnLeuPheHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.38"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      LysSerLysValGlyTrpLeuAlaGlnLeuPheHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.39"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      LysSerLysValGlyTrpLeuIleAlaLeuPheHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.40"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      LysSerLysValGlyTrpLeuIleGlnAlaPheHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.41"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      LysSerLysValGlyTrpLeuIleGlnLeuAlaHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.42"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      LysSerLysValGlyTrpLeuIleGlnLeuPheAlaLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.43"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      LysSerLysValGlyTrpLeuIleGlnLeuPheHisAlaLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.44"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      LysSerLysValGlyTrpLeuIleGlnLeuPheHisLysAla                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.56"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      IleLysIleSerGlyLysTrpLysAlaLysGlnArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.61"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      IleLysIleSerGlyLysPheLysAlaGlnLysArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.66"                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5..7                                                            (D) OTHER INFORMATION: /label= D- Trp                                         /note= "The amino acid at position 6 is                                       D- tryptophan"                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      IleLysIleSerGlyLysTrpLysAlaGlnLysArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.67"                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6..8                                                            (D) OTHER INFORMATION: /label= Substituted-Ala                                /note= "The alanine at position 7 is                                          beta-1- naphthyl-substituted"                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      IleLysIleSerGlyLysAlaLysAlaGlnLysArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.9"                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      GlnLysArgPheLeuLysLysTrpLysAlaGlnLysArgPheLeuLys                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.30"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      LysTrpLysAlaGlnLysArgPheLeuLysLysSerLysValGlyTrp                              151015                                                                        LeuIleGlnLeuPheHisLysLys                                                      20                                                                            (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.63"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      IleLysIleSerGlyLysTrpLysAlaGlnLysArgPheLeuLysLys                              151015                                                                        SerLysValGlyTrpLeuIleGlnLeuPheHisLysLys                                       2025                                                                          (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.7"                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      LysTrpLysAlaGlnLysArgPheLeuLysLeuTrpLysAlaGlnLys                              151015                                                                        ArgPheLeuLys                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.10.1"                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      LysArgPheLeuLysLysTrpLysAlaGlnLysArgPheLeuLysLys                              151015                                                                        TrpLysAlaGlnLysArgPheLeuLys                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.29"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      LysSerLysValGlyTrpLeuIleGlnLeuPheHisLysLysLysSer                              151015                                                                        LysValGlyTrpLeuIleGlnLeuPheHisLysLys                                          2025                                                                          (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.46"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      LysTrpLysAlaAlaAlaLysArgPheLeuLysLysTrpLysAlaGln                              151015                                                                        LysArgPheLeuLys                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.47"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      LysTrpLysAlaGlnLysArgPheLeuLysLysTrpLysAlaAlaAla                              151015                                                                        LysArgPheLeuLys                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.49"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      LysTrpLysAlaAlaAlaLysArgPheLeuLysLysTrpLysAlaAla                              151015                                                                        AlaLysArgPheLeu                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.69"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      LysTrpLysAlaAlaAlaArgPheLeuLysLysTrpLysAlaAlaAla                              151015                                                                        ArgPheLeuLysLysTrpLysAlaAlaAlaArgPheLeuLys                                    202530                                                                        (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.55"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      GlyTrpLeuIleGlnLeuPheHisLysLysIleGluMetAsnSer                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.73"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      IleLysIleSerGlyLysTrpLysAlaGlnPheArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.70"                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8..10                                                           (D) OTHER INFORMATION: /label= Substituted-Ala                                /note= "The alanine at position 9 is                                          beta-3- pyridyl-substituted"                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      IleLysIleSerGlyLysTrpLysAlaGlnLysArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:64:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.72"                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1..3                                                            (D) OTHER INFORMATION: /label= D- alanine                                     /note= "The position 1 and position 2 alanine                                 residues are both D-alanine"                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                      AlaAlaIleLysIleSerGlyLysTrpLysAlaGlnLysArgPheLeu                              151015                                                                        Lys                                                                           (2) INFORMATION FOR SEQ ID NO:65:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.10.2"                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                      GlnLysArgPheLeuLysLysTrpLysAlaGlnLysArgPheLeuLys                              151015                                                                        LysTrpLysAlaGlnLysArgPheLeuLys                                                2025                                                                          (2) INFORMATION FOR SEQ ID NO:66:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.71"                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 13..15                                                          (D) OTHER INFORMATION: /label= Substituted-Ala                                /note= "The alanine at position 13 is                                         beta-3- pyridyl-substituted"                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                      IleLysIleSerGlyLysTrpLysAlaGlnLysArgAlaLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:67:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.5"                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                      HisValHisIleSerLysSerLysValGlyTrpLeuIleGlnLeuPhe                              151015                                                                        HisLysLysIleGlu                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:68:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.65 reduced"                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 1..17                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                      CysIleLysIleSerGlyLysTrpLysAlaGlnLysArgPheLeuLys                              151015                                                                        Cys                                                                           (2) INFORMATION FOR SEQ ID NO:69:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 487 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "rBPI"                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                      MetArgGluAsnMetAlaArgGlyProCysAsnAlaProArgTrpVal                              31-30-25-20                                                                   SerLeuMetValLeuValAlaIleGlyThrAlaValThrAlaAlaVal                              15-10-51                                                                      AsnProGlyValValValArgIleSerGlnLysGlyLeuAspTyrAla                              51015                                                                         SerGlnGlnGlyThrAlaAlaLeuGlnLysGluLeuLysArgIleLys                              202530                                                                        IleProAspTyrSerAspSerPheLysIleLysHisLeuGlyLysGly                              354045                                                                        HisTyrSerPheTyrSerMetAspIleArgGluPheGlnLeuProSer                              50556065                                                                      SerGlnIleSerMetValProAsnValGlyLeuLysPheSerIleSer                              707580                                                                        AsnAlaAsnIleLysIleSerGlyLysTrpLysAlaGlnLysArgPhe                              859095                                                                        LeuLysMetSerGlyAsnPheAspLeuSerIleGluGlyMetSerIle                              100105110                                                                     SerAlaAspLeuLysLeuGlySerAsnProThrSerGlyLysProThr                              115120125                                                                     IleThrCysSerSerCysSerSerHisIleAsnSerValHisValHis                              130135140145                                                                  IleSerLysSerLysValGlyTrpLeuIleGlnLeuPheHisLysLys                              150155160                                                                     IleGluSerAlaLeuArgAsnLysMetAsnSerGlnValCysGluLys                              165170175                                                                     ValThrAsnSerValSerSerLysLeuGlnProTyrPheGlnThrLeu                              180185190                                                                     ProValMetThrLysIleAspSerValAlaGlyIleAsnTyrGlyLeu                              195200205                                                                     ValAlaProProAlaThrThrAlaGluThrLeuAspValGlnMetLys                              210215220225                                                                  GlyGluPheTyrSerGluAsnHisHisAsnProProProPheAlaPro                              230235240                                                                     ProValMetGluPheProAlaAlaHisAspArgMetValTyrLeuGly                              245250255                                                                     LeuSerAspTyrPhePheAsnThrAlaGlyLeuValTyrGlnGluAla                              260265270                                                                     GlyValLeuLysMetThrLeuArgAspAspMetIleProLysGluSer                              275280285                                                                     LysPheArgLeuThrThrLysPhePheGlyThrPheLeuProGluVal                              290295300305                                                                  AlaLysLysPheProAsnMetLysIleGlnIleHisValSerAlaSer                              310315320                                                                     ThrProProHisLeuSerValGlnProThrGlyLeuThrPheTyrPro                              325330335                                                                     AlaValAspValGlnAlaPheAlaValLeuProAsnSerSerLeuAla                              340345350                                                                     SerLeuPheLeuIleGlyMetHisThrThrGlySerMetGluValSer                              355360365                                                                     AlaGluSerAsnArgLeuValGlyGluLeuLysLeuAspArgLeuLeu                              370375380385                                                                  LeuGluLeuLysHisSerAsnIleGlyProPheProValGluLeuLeu                              390395400                                                                     GlnAspIleMetAsnTyrIleValProIleLeuValLeuProArgVal                              405410415                                                                     AsnGluLysLeuGlnLysGlyPheProLeuProThrProAlaArgVal                              420425430                                                                     GlnLeuTyrAsnValValLeuGlnProHisGlnAsnPheLeuLeuPhe                              435440445                                                                     GlyAlaAspValValTyrLys                                                         450455                                                                        (2) INFORMATION FOR SEQ ID NO:70:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.74"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                      LysSerLysValGlyTrpLeuIleGlnLeuPheHisLysLysLys                                 151015                                                                        TrpLysAlaGlnLysArgPheLeuLys                                                   20                                                                            (2) INFORMATION FOR SEQ ID NO:71:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.76"                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 10..12                                                          (D) OTHER INFORMATION: /label= D- Phe                                         /note= "The amino acid at position 6 is                                       D- phenylalanine"                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                      IleLysIleSerGlyLysTrpLysAlaGlnLysArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:72:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.77"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                      IleLysIleSerGlyLysTrpLysAlaGlnTrpArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:73:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.79"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                      IleLysIleSerGlyLysTrpLysAlaLysLysArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:74:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.80"                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 10..12                                                          (D) OTHER INFORMATION: /label= Substituted-Ala                                /note= "The alanine at position 11 is                                         beta-1- naphthyl-substituted"                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                                      IleLysIleSerGlyLysTrpLysAlaGlnAlaArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:75:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.81"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                                      IleLysIleSerGlyLysTrpLysAlaPheLysArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:76:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.82"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                                      LysSerLysValGlyTrpLeuIleGlnLeuTrpHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:77:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.83"                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 10..12                                                          (D) OTHER INFORMATION: /label= Substituted-Ala                                /note= "The alanine at position 11 is                                         beta-1- naphthyl-substituted"                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                                      LysSerLysValGlyTrpLeuIleGlnLeuAlaHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:78:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.84"                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6..8                                                            (D) OTHER INFORMATION: /label= Substituted-Ala                                /note= "The alanine at position 7 is                                          beta-1- naphthyl-substituted"                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                                      IleLysIleSerGlyLysAlaLysAlaGlnPheArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:79:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.85"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                                      LysSerLysValLeuTrpLeuIleGlnLeuPheHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:80:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.86"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                                      LysSerLysValGlyTrpLeuIleLeuLeuPheHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:81:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.87"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                                      LysSerLysValGlyTrpLeuIleGlnLeuPheLeuLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:82:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.88"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                                      IleLysIleSerGlyLysTrpLysAlaPhePheArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:83:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.98"                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6..8                                                            (D) OTHER INFORMATION: /label= Substituted-Ala                                /note= "The alanine at position 7 is                                          beta-1- naphthyl-substituted"                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                                      IleLysIleSerGlyLysAlaLysAlaPheLysArgPheLeuLys                                 151015                                                                        LysSerLysValGlyTrpLeuIleGlnLeuPheHisLysPhe                                    2025                                                                          (2) INFORMATION FOR SEQ ID NO:84:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.89"                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6..8                                                            (D) OTHER INFORMATION: /label= Substituted-Ala                                /note= "The alanine at position 7 is                                          beta-1- naphthyl-substituted"                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:                                      IleLysIleSerGlyLysAlaLysAlaPheLysArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:85:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.90"                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6..8                                                            (D) OTHER INFORMATION: /label= Substituted-Ala                                /note= "The alanine at position 7 is                                          beta-1- naphthyl-substituted"                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                                      IleLysIleSerGlyLysAlaLysAlaPhePheArgPheLeuLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:86:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.91"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                                      LysSerLysValGlyTrpLeuIlePheLeuPheHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:87:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.92"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:                                      LysSerLysValGlyTrpLeuIleLysLeuPheHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:88:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.93"                                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6..8                                                            (D) OTHER INFORMATION: /label= Substituted-Ala                                /note= "The alanine at position 7 is                                          beta-1- naphthyl-substituted"                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:                                      IleLysIleSerGlyLysAlaLysAlaGlnPheArgPheLeuLys                                 151015                                                                        LysSerLysValGlyTrpLeuIleGlnLeuPheHisLysLys                                    2025                                                                          (2) INFORMATION FOR SEQ ID NO:89:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.94"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:                                      LysSerLysValGlyTrpLeuIleGlnLeuPhePheLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:90:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.95"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:                                      LysSerLysValPheTrpLeuIleGlnLeuPheHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:91:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.96"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:                                      LysSerLysValGlyTrpLeuIleGlnLeuPheHisLysPhe                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:92:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.97"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:                                      LysSerLysValLysTrpLeuIleGlnLeuPheHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:93:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.99"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:                                      LysTrpLysAlaGlnTrpArgPheLeuLysLysTrpLysAlaGln                                 151015                                                                        TrpArgPheLeuLysLysTrpLysAlaGlnTrpArgPheLeuLys                                 202530                                                                        (2) INFORMATION FOR SEQ ID NO:94:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.100"                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:                                      LysSerLysValLysTrpLeuIleLysLeuPheHisLysLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:95:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (D) OTHER INFORMATION: "BPI.101"                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:                                      LysSerLysValLysTrpLeuIleLysLeuPhePheLysPheLysSer                              151015                                                                        LysValLysTrpLeuIleLysLeuPhePheLysPhe                                          2025                                                                          (2) INFORMATION FOR SEQ ID NO:96:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: /note= "BPI.102"                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:                                      LysTrpLysAlaGlnPheArgPheLeuLysLysSerLysValGlyTrp                              151015                                                                        LeuIleLeuLeuPheHisLysLys                                                      20                                                                            (2) INFORMATION FOR SEQ ID NO:97:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1443 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1443                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 76..1443                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..1443                                                         (D) OTHER INFORMATION: /note= "rLBP"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:                                      ATGGGGGCCTTGGCCAGAGCCCTGCCGTCCATACTGCTGGCATTGCTG48                            MetGlyAlaLeuAlaArgAlaLeuProSerIleLeuLeuAlaLeuLeu                              25-20-15-10                                                                   CTTACGTCCACCCCAGAGGCTCTGGGTGCCAACCCCGGCTTGGTCGCC96                            LeuThrSerThrProGluAlaLeuGlyAlaAsnProGlyLeuValAla                              515                                                                           AGGATCACCGACAAGGGACTGCAGTATGCGGCCCAGGAGGGGCTATTG144                           ArgIleThrAspLysGlyLeuGlnTyrAlaAlaGlnGluGlyLeuLeu                              101520                                                                        GCTCTGCAGAGTGAGCTGCTCAGGATCACGCTGCCTGACTTCACCGGG192                           AlaLeuGlnSerGluLeuLeuArgIleThrLeuProAspPheThrGly                              253035                                                                        GACTTGAGGATCCCCCACGTCGGCCGTGGGCGCTATGAGTTCCACAGC240                           AspLeuArgIleProHisValGlyArgGlyArgTyrGluPheHisSer                              40455055                                                                      CTGAACATCCACAGCTGTGAGCTGCTTCACTCTGCGCTGAGGCCTGTC288                           LeuAsnIleHisSerCysGluLeuLeuHisSerAlaLeuArgProVal                              606570                                                                        CCTGGCCAGGGCCTGAGTCTCAGCATCTCCGACTCCTCCATCCGGGTC336                           ProGlyGlnGlyLeuSerLeuSerIleSerAspSerSerIleArgVal                              758085                                                                        CAGGGCAGGTGGAAGGTGCGCAAGTCATTCTTCAAACTACAGGGCTCC384                           GlnGlyArgTrpLysValArgLysSerPhePheLysLeuGlnGlySer                              9095100                                                                       TTTGATGTCAGTGTCAAGGGCATCAGCATTTCGGTCAACCTCCTGTTG432                           PheAspValSerValLysGlyIleSerIleSerValAsnLeuLeuLeu                              105110115                                                                     GGCAGCGAGTCCTCCGGGAGGCCCACAGTTACTGCCTCCAGCTGCAGC480                           GlySerGluSerSerGlyArgProThrValThrAlaSerSerCysSer                              120125130135                                                                  AGTGACATCGCTGACGTGGAGGTGGACATGTCGGGAGACTTGGGGTGG528                           SerAspIleAlaAspValGluValAspMetSerGlyAspLeuGlyTrp                              140145150                                                                     CTGTTGAACCTCTTCCACAACCAGATTGAGTCCAAGTTCCAGAAAGTA576                           LeuLeuAsnLeuPheHisAsnGlnIleGluSerLysPheGlnLysVal                              155160165                                                                     CTGGAGAGCAGGATTTGCGAAATGATCCAGAAATCGGTGTCCTCCGAT624                           LeuGluSerArgIleCysGluMetIleGlnLysSerValSerSerAsp                              170175180                                                                     CTACAGCCTTATCTCCAAACTCTGCCAGTTACAACAGAGATTGACAGT672                           LeuGlnProTyrLeuGlnThrLeuProValThrThrGluIleAspSer                              185190195                                                                     TTCGCCGACATTGATTATAGCTTAGTGGAAGCCCCTCGGGCAACAGCC720                           PheAlaAspIleAspTyrSerLeuValGluAlaProArgAlaThrAla                              200205210215                                                                  CAGATGCTGGAGGTGATGTTTAAGGGTGAAATCTTTCATCGTAACCAC768                           GlnMetLeuGluValMetPheLysGlyGluIlePheHisArgAsnHis                              220225230                                                                     CGTTCTCCAGTTACCCTCCTTGCTGCAGTCATGAGCCTTCCTGAGGAA816                           ArgSerProValThrLeuLeuAlaAlaValMetSerLeuProGluGlu                              235240245                                                                     CACAACAAAATGGTCTACTTTGCCATCTCGGATTATGTCTTCAACACG864                           HisAsnLysMetValTyrPheAlaIleSerAspTyrValPheAsnThr                              250255260                                                                     GCCAGCCTGGTTTATCATGAGGAAGGATATCTGAACTTCTCCATCACA912                           AlaSerLeuValTyrHisGluGluGlyTyrLeuAsnPheSerIleThr                              265270275                                                                     GATGAGATGATACCGCCTGACTCTAATATCCGACTGACCACCAAGTCC960                           AspGluMetIleProProAspSerAsnIleArgLeuThrThrLysSer                              280285290295                                                                  TTCCGACCCTTCGTCCCACGGTTAGCCAGGCTCTACCCCAACATGAAC1008                          PheArgProPheValProArgLeuAlaArgLeuTyrProAsnMetAsn                              300305310                                                                     CTGGAACTCCAGGGATCAGTGCCCTCTGCTCCGCTCCTGAACTTCAGC1056                          LeuGluLeuGlnGlySerValProSerAlaProLeuLeuAsnPheSer                              315320325                                                                     CCTGGGAATCTGTCTGTGGACCCCTATATGGAGATAGATGCCTTTGTG1104                          ProGlyAsnLeuSerValAspProTyrMetGluIleAspAlaPheVal                              330335340                                                                     CTCCTGCCCAGCTCCAGCAAGGAGCCTGTCTTCCGGCTCAGTGTGGCC1152                          LeuLeuProSerSerSerLysGluProValPheArgLeuSerValAla                              345350355                                                                     ACTAATGTGTCCGCCACCTTGACCTTCAATACCAGCAAGATCACTGGG1200                          ThrAsnValSerAlaThrLeuThrPheAsnThrSerLysIleThrGly                              360365370375                                                                  TTCCTGAAGCCAGGAAAGGTAAAAGTGGAACTGAAAGAATCCAAAGTT1248                          PheLeuLysProGlyLysValLysValGluLeuLysGluSerLysVal                              380385390                                                                     GGACTATTCAATGCAGAGCTGTTGGAAGCGCTCCTCAACTATTACATC1296                          GlyLeuPheAsnAlaGluLeuLeuGluAlaLeuLeuAsnTyrTyrIle                              395400405                                                                     CTTAACACCTTCTACCCCAGGTTCAATGATAAGTTGGCCGAAGGCTTC1344                          LeuAsnThrPheTyrProArgPheAsnAspLysLeuAlaGluGlyPhe                              410415420                                                                     CCCCTTCCTCTGCTGAAGCGTGTTCAGCTCTACGACCTTGGGCTGCAG1392                          ProLeuProLeuLeuLysArgValGlnLeuTyrAspLeuGlyLeuGln                              425430435                                                                     ATCCATAAGGACTTCCTGTTCTTGGGTGCCAATGTCCAATACATGAGA1440                          IleHisLysAspPheLeuPheLeuGlyAlaAsnValGlnTyrMetArg                              440445450455                                                                  GTT1443                                                                       Val                                                                           (2) INFORMATION FOR SEQ ID NO:98:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 481 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:                                      MetGlyAlaLeuAlaArgAlaLeuProSerIleLeuLeuAlaLeuLeu                              25-20-15-10                                                                   LeuThrSerThrProGluAlaLeuGlyAlaAsnProGlyLeuValAla                              515                                                                           ArgIleThrAspLysGlyLeuGlnTyrAlaAlaGlnGluGlyLeuLeu                              101520                                                                        AlaLeuGlnSerGluLeuLeuArgIleThrLeuProAspPheThrGly                              253035                                                                        AspLeuArgIleProHisValGlyArgGlyArgTyrGluPheHisSer                              40455055                                                                      LeuAsnIleHisSerCysGluLeuLeuHisSerAlaLeuArgProVal                              606570                                                                        ProGlyGlnGlyLeuSerLeuSerIleSerAspSerSerIleArgVal                              758085                                                                        GlnGlyArgTrpLysValArgLysSerPhePheLysLeuGlnGlySer                              9095100                                                                       PheAspValSerValLysGlyIleSerIleSerValAsnLeuLeuLeu                              105110115                                                                     GlySerGluSerSerGlyArgProThrValThrAlaSerSerCysSer                              120125130135                                                                  SerAspIleAlaAspValGluValAspMetSerGlyAspLeuGlyTrp                              140145150                                                                     LeuLeuAsnLeuPheHisAsnGlnIleGluSerLysPheGlnLysVal                              155160165                                                                     LeuGluSerArgIleCysGluMetIleGlnLysSerValSerSerAsp                              170175180                                                                     LeuGlnProTyrLeuGlnThrLeuProValThrThrGluIleAspSer                              185190195                                                                     PheAlaAspIleAspTyrSerLeuValGluAlaProArgAlaThrAla                              200205210215                                                                  GlnMetLeuGluValMetPheLysGlyGluIlePheHisArgAsnHis                              220225230                                                                     ArgSerProValThrLeuLeuAlaAlaValMetSerLeuProGluGlu                              235240245                                                                     HisAsnLysMetValTyrPheAlaIleSerAspTyrValPheAsnThr                              250255260                                                                     AlaSerLeuValTyrHisGluGluGlyTyrLeuAsnPheSerIleThr                              265270275                                                                     AspGluMetIleProProAspSerAsnIleArgLeuThrThrLysSer                              280285290295                                                                  PheArgProPheValProArgLeuAlaArgLeuTyrProAsnMetAsn                              300305310                                                                     LeuGluLeuGlnGlySerValProSerAlaProLeuLeuAsnPheSer                              315320325                                                                     ProGlyAsnLeuSerValAspProTyrMetGluIleAspAlaPheVal                              330335340                                                                     LeuLeuProSerSerSerLysGluProValPheArgLeuSerValAla                              345350355                                                                     ThrAsnValSerAlaThrLeuThrPheAsnThrSerLysIleThrGly                              360365370375                                                                  PheLeuLysProGlyLysValLysValGluLeuLysGluSerLysVal                              380385390                                                                     GlyLeuPheAsnAlaGluLeuLeuGluAlaLeuLeuAsnTyrTyrIle                              395400405                                                                     LeuAsnThrPheTyrProArgPheAsnAspLysLeuAlaGluGlyPhe                              410415420                                                                     ProLeuProLeuLeuLysArgValGlnLeuTyrAspLeuGlyLeuGln                              425430435                                                                     IleHisLysAspPheLeuPheLeuGlyAlaAsnValGlnTyrMetArg                              440445450455                                                                  Val                                                                           __________________________________________________________________________

What is claimed is:
 1. A peptide which has an amino acid sequence ofhuman bactericidal permeability/permeability-increasing protein (BPI)from about position 17 to about position 45 of SEQ ID NO: 69 andvariants of the sequence, having a biological activity that is anactivity of BPI.
 2. A peptide which contains two or three of the same ordifferent peptides according to claim 1 covalently linked together.
 3. Apeptide which has an amino acid sequence of humanbactericidal/permeability-increasing protein (BPI) from about position65 to about position 99 of SEQ ID NO: 69 and variants of the sequencehaving a biological activity that is an activity of BPI.
 4. A peptidewhich contains two or three of the same or different peptides accordingto claim 3 covalently linked together.
 5. A peptide which has an aminoacid sequence of human bactericidal/permeability-increasing protein(BPI) from about position 142 to about position 169 of SEQ ID NO:69 andvariants of the sequence having a biological activity that is anactivity of BPI.
 6. A peptide which contains two or three of the same ordifferent peptides according to claim 5 covalently linked together.
 7. Apeptide in which two or three of the same or different peptidesaccording to claims 1, 3 or 5 are directly covalently linked together.